RAC-PK as a therapeutic agent or in diagnostics, screening method for agents and process for activating RAC-PK

ABSTRACT

The invention concerns RAC-PK and fragments thereof, as well as activators and inhibitors of RAC-PK for use as medicaments, particularly in the treatment of diseases concerned with abnormalities in processes modulated by insulin, such as cellular proliferation, insulin deficiency and/or excess blood sugar levels. Moreover, the invention provides RAC-PK for use in screening potential mimics or modulators thereof. A method for screening for agents capable of affecting the activity of GSK3 is also disclosed. The invention further provides a screening kit comprising the RAC-PK as an active principle, and a method for screening compounds which are candidate mimics or modulators of RAC-PK activity comprising detecting specific interactions between the candidate compounds and RAC-PK. There is also provided a process for activating RAC-PK comprising treatment thereof with a phosphatase inhibitor.

This application is a Continuation In Part of application (I) Ser.No.10/673,091, Filing Date Sep. 26, 2003, pending, which is acontinuation of Ser. No. 09/845,667, filed Apr. 30, 2001, now abandoned,which is a continuation of Ser. No. 09/091,763, filed Jun. 19, 1998, nowabandoned, which is a National Stage of PCT/GB96/03186, filed Dec. 20,1996; (II) of application Ser. No.10/147,123, filed May 16, 2002, whichis a continuation of Ser. No. 09/542,646, filed Apr. 3, 2000, nowabandoned, which is a continuation of Ser. No. 09/091,109, filed Jun.11, 1998, now abandoned, which is a National Stage of PCT/EP96/0481 1,filed November 5, 1996; and (III) of application Ser. No. 09/970,000,pending, which is a Continuation of Ser. No. 09/068,702, filed May 13,1998, now abandoned, which is a National Stage of PCT/EP96/04810, filedNov. 5, 1996.

The present invention relates to the control of glycogen metabolism andprotein synthesis, in particular through the use of insulin.Particularly, the present invention is related to the use of RAC proteinkinase (RAC-PK) as a therapeutic agent, as a ligand for screeningmolecules for a possible interaction with RAC-PK and to a method foridentifying molecules involved in signal transduction. Further, thepresent invention relates to a method for producing an active form of akinase involved in an insulin-dependent signalling pathway.

BACKGROUND OF THE INVENTION

Many people with diabetes have normal levels of insulin in their blood,but the insulin fails to stimulate muscle cells and fat cells in thenormal way (type II diabetes). Currently it is believed that there is abreakdown in the mechanism through which insulin signals to the muscleand fat cells.

Protein phosphorylation and dephosphorylation are fundamental processesfor the regulation of cellular functions. Protein phosphorylation isprominently involved in signal transduction, where extracellular signalsare propagated and amplified by a cascade of protein phosphorylation anddephosphorylation. Two of the best characterized signal transduction,where extracellular signals are propagated and amplified by a cascade ofprotein phosphorylation and dephosphorylation. Two of the bestcharacterized signal transduction pathways involve the c-AMP-dependantprotein kinase (PKA) and protein kinase C (PKC). Each pathway uses adifferent second messenger molecule to activate the protein kinase,which, in turn, phosphorylates specific target molecules.

A novel subfamily of serine (Ser)/threonine (Thr) kinases has beenrecently identified and cloned, termed herein the RAC-PK [see Jones etal., Proc Natl Acad Sci USA, Vol. 88, No. 10, pp. 4171-4175 (1991); andJones, Jakubowicz and Hemmings, Cell Regul, Vol. 2, No. 12, pp. 1001-1009 (1991)], but also known as RAC-PK or Akt. RAC kinases have beenidentified in two closely-related isoforms, RACα and RACβ, which share90% homology at the gene sequence. Mouse RACα (c-akt) is the cellularhomologue of the viral oncogene v-akt, generated by fusion of the Gagprotein from the AKT8 retrovirus to the N-terminus of murine c-akt.Human RACβ is found to be involved in approximately 10% of ovariancarcinomas, suggesting an involvement of RAC kinases in cell growthregulation.

Another kinase implicated in cell growth control is S6 kinase, known asp70S6K. S6 kinase phosphorylates the 40S ribosomal protein S6, an eventwhich up-regulates protein synthesis and is believed to be required inorder for progression through the G1 phase of the cell cycle. Theactivity of p70S6K is regulated by Ser/Thr phosphorylation thereof, andit is itself a Ser/Thr kinase. The p70S6K signaling pathway is believedto consist of a series of Ser/Thr kinases, activating each other in turnand leading to a variety of effects associated with cell proliferationand growth. RAC-PK is believed to lie on the same signaling pathway asp70S6K, but upstream thereof.

RAC kinases contain an amino-terminal pleckstrin homology (PH) domain.See Haslam, Koide and Hemmings, Nature, Vol. 363, No. 6427, pp. 309-310(1993). The PH domain was originally identified as an internal repeat,present at the amino and carboxy-termini of pleckstrin, a 47 kDa proteinwhich is the major PKC substrate in activated platelets. See Tyers etal., Nature, Vol. 333, No. 6172, pp. 470-473 (1988). The superfamily ofPH domain containing molecules consists of over 90 members includingSer/Thr kinases, e.g., RAC, Nrk, β-adrenergic receptor kinase (βARK) andPKCμ; tyrosine kinases, e.g., Bruton's tyrosine kinase (Btk), Tec andItk; GTPase regulators, e.g., ras-GAP, ras-GRF, Vav, SOS and BCR; allknown mammalian phospholipase Cs; cytoskeletal proteins, e.g.,β-spectrin, AFAP-110 and syntrophin; “adapter” proteins, e.g., GRB-7 and3BP2; and kinase substrates, e.g., pleckstrin and IRS-1.

While the PH domain structure has been solved for β-spectrin, dynaminand pleckstrin's amino-terminal domain, its precise function remainsunclear. The presence of PH domains in many signaling and cytoskeletalproteins implicates it in mediating protein-protein and membraneinteractions. Indeed, the PH domain of the βARK appears partlyresponsible for its binding to the βγ-subunits of the heterotrimericG-proteins associated with the β-adrenergic receptor, while the PHdomain of the Btk appears to mediate an interaction with PKC. Several PHdomains have been shown to be able to bindphosphatidyl-inositol-4-5-bisphosphate in vitro, although weakly.

IMPDH is a highly-conserved enzyme (41% amino acid identity betweenbacterial and mammalian sequences) involved in the rate-limiting step ofguanine biosynthesis. In mammals there are two isoforms, 84% identical,called type I and type II which are differentially-expressed. SeeNatsumeda et al., J Biol Chem, Vol. 265, No. 9, pp. 5292-5295 (1990).Type I is constitutively-expressed at low levels while the type II mRNAand protein levels increase during cellular proliferation. IMPDHactivity levels are also elevated during rapid proliferation in manycells. See Collart and Huberman, J Biol Chem, Vol. 263, No. 30, pp.15769-15772 (1988).

By measuring the metabolic fluxes, the proliferative index of intactcancer cells has been shown to be linked with the preferentialchannelling of IMP into guanylate biosynthesis. Inhibition of cellularIMPDH activity results in an abrupt cessation of DNA synthesis and acell-cycle block at the G₁-S interface. The specific inhibition of IMPDHby tiazofurin and the subsequent decline in the GTP pool, results in thedown regulation of the G-protein ras, which is involved in many signaltransduction pathways leading to cellular proliferation. For review seeAvruch, Zhang and Kyriakis, Trends Biochem Sci, Vol. 19, No. 7, pp.279-283 (1994).

Interestingly, p53 has been implicated in regulating IMPDH activitylevels. See Sherley, J Biol Chem, Vol. 266, No. 36, pp. 24815-24828(1991). Here a moderate over-expression of p53 (3- to 6-fold) induces aprofound growth arrest which is rescued by purine nucleotide precursors.Indeed, the p53 over-expression induces a specific block in IMP to XMPconversion, and a diminished activity level of IMPDH. The p53 block doesnot affect the rate of RNA synthesis, nor is the phenotype rescued bydeoxynucleotides indicating that a lack of precursors for DNA synthesisis also not the cause of the block. It would seem most likely that thiseffect is mediated through a down-regulation of the GTP pool required byG-proteins, such as ras.

The above observations suggest that IMPDH type II is primarily involvedin producing XMP which is channelled into the GTP pool which is crucialfor the regulation of G-proteins involved in signal transduction, suchas ras. It may be that the type I enzyme provides a basal level of XMPthat is channelled into the GTP/dGTP pools required for RNA and DNAsynthesis. Changes in IMPDH type II activity would alter the GTP/GDPratio by specifically altering the GTP component which could greatlyaffect ras signalling pathways as ras is sensitive to small changes inthe GTP/GDP ratio.

Glycogen synthase kinase-3 (GSK3) is implicated in the control ofseveral processes important for mammalian cell physiology, includingglycogen metabolism and the control of protein synthesis by insulin, aswell as the modulation of activity of several transcription factors,such as AP-1 and CREB. GSK3 is inhibited in vitro by serinephosphorylation caused by MAP kinase and p70^(S6K), kinases which lie ondistinct insulin-stimulated signalling pathways.

GSK3 is responsible for serine phosphorylation in glycogen synthase,whose dephosphorylation underlies the stimulation of glycogen synthesisby muscle. Thus, GSK3 inactivates glycogen synthase, resulting in anincrease in blood sugar levels. Insulin inhibits the action of GSK3,which, in combination with the concomitant activation of phosphataseswhich dephosphory late glycogen synthase, leads to the activation ofglycogen synthase and the lowering of blood sugar levels.

GSK3 is inhibited in response to insulin with a half-time of 2 minutes,slightly slower than the half-time for activation of RAC-PKα (1 minute).Inhibition of GSK3 by insulin results in its phosphorylation at the sameserine residue (serine 21) which is targeted by RAC-PKα in vitro. Likethe activation of RAC-PKα, the inhibition of GSK3 by insulin isprevented by phosphatidyl inositol (PI-3) kinase inhibitors wortmanninand LY 294002. The inhibition of GSK3 is likely to contribute to theincrease in the rate of glycogen synthesis [see Cross et al., Biochem J,Vol. 303, Pt. 1, pp. 21-26 (1994)] and translation of certain mRNAs byinsulin. See Welsh et al., Biochem J, Vol. 303, Pt. 1, pp. 15-20 (1994).

We have used the yeast two-hybrid system [see Fields and Song, Nature,Vol. 340, No. 6230, pp. 245-246 (1989); and Chien, Bartel, Sternglanzand Fields, Proc Natl Acad Sci USA, Vol. 88, No. 21, pp. 9578-9582(1991)] to determine if RAC-PK could function by forming specificinteractions with other proteins. We have identified RAC-PK asinteracting with human inosine-5′ monophosphate dehydrogenase (IMPDH)type II, and with a novel protein termed RAC-PK Carboxy-Terminal BindingProtein (CTBP). RAC-PK stimulates IMPDH type II activity. In conjunctionwith the known role of IMPDH in GTP biosynthesis, our findings suggest arole for RAC-PK in the regulation of cell proliferation.

Moreover, using a peptide derived from GSK3 and GSK3 itself, we havebeen able to show that RAC-PK interacts with, phosphorylates andinactivates GSK3. This implicates RAC-PK in the regulation ofinsulin-dependent signalling pathways, which control cellularproliferation. Taken together, these results suggest a major involvementfor RAC-PK in the control of insulin action.

Many growth factors trigger the activation of phosphatidylinositol (PI)3-kinase, the enzyme which converts PI 4,5 bisphosphate (PIP2) to theputative second messenger PI 3,4,5 trisphosphate (PIP3) and RAC-PK liesdownstream of PI 3-kinase. See Franke et al., Cell, Vol. 81, No. 5, pp.727-736 (1995). RAC-PKα is converted from an inactive to an active formwith a half-time of about 1 minute when cells are stimulated with PDGF[see Franke et al. (1995), supra], EGF or basic FGF [see Burgering andCoffer, Nature, Vol. 376, No. 6541, pp. 599-602 (1995)] or insulin [seeCross et al. (1995), supra; and Kohn, Kovacina and Roth, EMBO J, Vol.14,No. 17, pp. 4288-4295 (1995)] or perpervanadate. See Andjelkovic et al.,Proc Natl Acad Sci USA, Vol. 93, No. 12, pp. 5699-5704 (1996).Activation of RAC-PK by insulin or growth factors is prevented if thecells are pre-incubated with inhibitors of PI 3-kinase (wortmannin or LY294002) or by over-expression of a dominant negative mutant of PI3-kinase. See Burgering and Coffer (1995), supra. Mutation of thetyrosine residues in the PDGF receptor that when phosphorylated bind toPI 3-kinase also prevent the activation of RAC-PKα. See Burgering andCoffer (1995), supra; and Franke et al. (1995), supra.

When isolated from natural sources, especially convenient sources, suchas tissue culture cells, RAC-PK and other signaling kinases are normallyin the inactive state. In order to isolate active PKs, it is necessaryto stimulate cells in order to switch on the signaling pathway to yieldactive kinase. Moreover, when cells expressing kinase enzymes are usedin kinase activity assays, it is necessary to employ activating agentsprior to conducting the assay. Thus, cells are normally stimulated withmitogens and/or activating agents, such as IL-2, platelet-derived growthfactor (PDGF), insulin, epidermal growth factor (EGF) and basicfibroblast growth factor (bFGF). Such agents are expensive and, when itis desired to produce active kinases or to activate cells in largeamounts, the use of such agents is disadvantageous.

Screening of candidate compounds for activity as inhibitors of RAC-PK,or other signaling kinases in order to identify candidateimmunosuppressive or anti-proliferative agents requires a plentifulsupply of PK. Using modern day technology, it is possible to producelarge quantities of virtually any desired protein in recombinant DNAexpression systems. In the case of kinases, such as those with which weare presently concerned, however, such systems are unsatisfactorybecause the proteins produced would be unphosphorylated and thereforeinactive. There is therefore a requirement to identify a cost-effectiveway to produce phosphorylated PKs which can be employed in screeningprocedures.

It is known [see Janö et al., Biochemistry, Vol. 85, pp. 406-410 (1988)]that vanadate can activate p70S6K itself. The mechanism of thisactivation, however, is not known. We have now found that vanadate actsgenerally on signaling kinases, activating them and preventingdeactivation by phosphatases. Moreover, we have found that okadaic acid,a different class of compound from vanadate which interacts withdifferent proteins, may be used to similar effect.

SUMMARY OF THE INVENTION

According to the invention, there is provided RAC-PK and fragments,analogues, isoforms and functional equivalents thereof, as well asactivators and inhibitors of RAC-PK for use in the treatment of diseasesconcerned with abnormalities in processes modulated by insulin, such ascellular proliferation, insulin deficiency and/or excess blood sugarlevels, e.g., in the treatment of type II diabetes and cancer, such asovarian, breast and pancreatic cancer. Moreover, the invention providesRAC-PK for use in screening potential mimics or modulators thereof. Theinvention further provides a screening kit comprising the RAC-PK as anactive principle, and a method for screening compounds which arecandidate mimics or modulators of RAC-PK activity comprising detectingspecific interactions between the candidate compounds and RAC-PK.

The present invention also provides a novel peptide comprising the aminoacid sequence Arg-xaa-Arg-Yaa-zaa-Ser/Thr-Hyd, where Xaa is any aminoacid, Yaa and Zaa are any amino acid [preferably not glycine (Gly)] andHyd is a large hydrophobic residue, such as Phe or Leu, or a functionalequivalent thereof. The invention also provides a method for screeningfor substances which inhibit the activation of RAC-PK in vivo bypreventing its interaction with PIP3 or P13,4-bisP. Thus the inventionalso provides a method of determining the ability of a substance toaffect the activity or activation of RAC-PK. The method of the inventioncan also be used for identifying activators or inhibitors of GSK3. Theinvention also provides a method for screening for inhibitors oractivators of enzymes that catalyse the phosphorylation of RAC-PK.

There is also provided a process for producing an active form of akinase involved in an insulin depedent signalling pathway.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect, the invention provides RAC-PK or a fragment thereof,or a modulator thereof except vanadate and wortmannin, for use as amedicament.

Vanadate, which term as used herein includes various forms thereof, suchas ortho- and metavanadate, pervanadate and other related vanadium ions,is known as a therapeutic agent in the treatment of diabetes. See, e.g.,U.S. Pat. No. 5,421,125, European Patent Application Nos. 0521787,0264278 and 0245979. In UK Patent Application No. 9525702.8 (Ciba-GeigyAG), filed Dec. 15, 1995, it is disclosed that vanadate is a potentactivator of kinases of the insulin-stimulated signalling pathways andof RAC-PK in particular. Accordingly vanadate exerts its therapeuticeffect on diabetes by stimulating RAC, which phosphorylates GSK3 anddeactivates it, leading to a lowering in blood sugar levels.

RAC-PK and activators thereof are therefore useful in the treatment ofdiabetes and other diseases where blood sugar levels are excessive.Conversely, inhibitors of RAC-PK, such as wortmannin and okadaic acidare useful in the treatment of diseases involving insufficiency in bloodsugar levels.

In the present invention, RAC-PK may be any isoform of RAC-PK asdescribed in the literature, from any species. Human RAC-PK ispreferred. Human RAC-PKα is represented in SEQ ID NO. 3. Domains ofRAC-PK are the individual functional portions thereof, such as the PHdomain, the catalytic domain and the C-terminal domain. Fragments ofRAC, which include domains of RAC, are functionally-active portions ofthe RAC-PK which may be used in the present invention in place of RAC.Fragments of RAC-PK are preferably the domains thereof, advantageouslythe PH domain, the catalytic domain and the C-terminal domain. In eachcase, the terminology used embraces mutants and derivatives of RAC-PKand its fragments which can be created or derived from thenaturally-occurring protein according to available technology. Forinstance, nucleic acids encoding RAC-PK may be mutated without affectingthe nature of the peptide encoded thereby, according to the degeneracyof the amino acid code. Moreover, conservative amino acid substitutionsmay be made in RAC-PK or a fragment thereof, substantially withoutaltering its function. Further additions, deletions and/or substitutionswhich improve or otherwise alter the function of RAC-PK or a fragmentthereof are envisaged and included within the scope of the invention.

The invention also provides the use of RAC-PK or a fragment thereof, ora modulator of RAC-PK activity, except vanadate, for the preparation ofa medicament for use in the treatment of diseases involving an anomalyin blood sugar levels, such as diabetes.

Moreover, the invention provides the use of RAC-PK or a fragmentthereof, or a modulator of RAC-PK activity, except wortmannin, for thepreparation of a medicament for use in the treatment of abnormalities incellular proliferation.

The antibiotic wortmannin, which is known to inhibitphosphatidylinositol 3-OH kinase (PI-3K) activation, targets signaltransduction and indirectly inactivates inter alia RAC, possibly viaPI-3K. Wortmannin has been indicated in the treatment of neoplasticconditions. However, the broad involvement of the various isoforms ofRAC-PK in mitogenic signal transduction, as well as insulin-dependentsignalling has hitherto not been known. We have now shown that RAC-PK isinvolved in the regulation of both GSK3 and IMPDH, a factor involved ingrowth control. It can be concluded, therefore, that RAC-PK plays acentral role in growth control.

Therapeutic agents according to the invention may be formulatedconventionally, according to the type of agent. Where the agent is asalt, such as vanadate, it is conveniently formulated in aqueoussolution at neutral pH and administered orally at room temperature. Inthe case of a peptide medicament, such as RAC-PK itself, more elaboratedelivery techniques, such as liposomal delivery, may be required inorder to introduce the peptide into target cells. Delivery systems forpeptide therapeutics are documented in the art.

The identification of RAC-PK as a major mediator in growth controlpermits the design of screening systems to identify putative therapeuticagents for use in treating anomalies of growth control. Thus, in asecond aspect of the invention there is provided a method for screeningpotential modulators of intracellular signalling comprising the stepsof:

-   -   (a) incubating RAC-PK or a fragment thereof with the compound to        be screened; and    -   (b) detecting interaction between the compound and RAC.

The screening may be carried out using complete RAC-PK or a fragmentthereof. In particular, it has been shown that the PH domain of RAC-PKis important in mediating many of its effects, as set out, e.g., in UKpatent application No. 9525703.6 (Ciba-Geigy AG), filed Dec. 15, 1995.Moreover, as disclosed hereinbelow, RAC-PK interacts with IMPDH via thePH domain. The interaction is not observable in the yeast two-hybridsystem if complete RAC-PK is used, although in vitro binding of RAC-PKto IMPDH occurs.

Interactions also occur between other fragments of RAC-PK and itsphysiological targets and regulators. For example, GSK3 binds to RAC-PKvia the catalytic domain, as evidenced by the phosphorylation of GSK3 byRAC. CTBP, on the other hand, does not bind the catalytic or PH domainsbut binds specifically to the carboxy terminal domain of RAC.

Preferably, therefore, the invention includes incubating the compound tobe screened with a fragment of RAC, which is advantageously the PHdomain, the catalytic domain or the carboxy terminal domain.

RAC-PK fragments for use in the method of the present invention may bein the form of isolated fragments, or in the form of the fragmentcomplexed with further polypeptides. For example, in the case of thetwo-hybrid system, the fragment is complexed to a DNA binding ortranscriptional activation domain derived from another protein, such asthe yeast activator GAL4.

Moreover, the RAC-PK used in the method of the invention may be in theform of a mutant thereof, e.g., a constitutively activated kinase. Animportant activating residue is T308, present in the so-called T-loopbetween subdomains 7 and 8 of the kinase. A general guide to kinasestructure is given in Woodgeft, Protein Kinases, IRL Press, UK (1994).Substitution of T308 with aspartic acid results in a clear increase inbasal activity of the kinase, which however retains a potential forfurther activation. The invention therefore provides a RAC-PK in whichThr308 has been mutated to Asp.

Preferably, Ser473 is additionally mutated to Asp. Phosphorylation ofthis residue is required for full activation of RAC-PK in vivo, and theT308/S473 double mutant (both residues converted to Asp) shows aconstitutive activity 18-fold higher than native RAC-PK. The doublemutant is not susceptible to further activation.

The mutations may be carried out by means of any suitable technique.Preferred, however, is in vitro site-directed mutagenesis of anucleotide sequence encoding RAC and subsequent expression of RAC in arecombinant DNA expression system. This method is an in vitromutagenesis procedure by which a defined site within a region of clonedDNA can be altered. See Zoller and Smith, Methods Enzymol, Vol. 100, pp.468-500 (1983); and Botstein and Shortle, Science, Vol. 229, No. 4719,pp. 1193-1201 (1985). Methods for site-directed mutagenesis arewell-known to those of skill in the art, as exemplified by Sambrook etal., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor, N.Y.,USA (1989), and the number of commercially-available in vitromutagenesis kits.

The compound to be screened may be present in essentially pure,uncomplexed form, or may be complexed with chemical groups or furtherpolypeptides. In the case of the two hybrid system, it is complexed to aDNA binding or transcriptional activation domain, in order to complementthe PH domain.

Isolated PH domain for use in the present invention may be prepared asset forth in UK patent application No. 9525705.1 (Ciba-Geigy AG), filedDec. 15, 1995. Where a small quantity of PH domain suffices, however, PHdomain may be obtained by expressing a nucleic acid sequence encoding itin bacterial cell culture in the form of a fusion protein which issubsequently cleaved according to techniques known in the art. Forexample, amino acids 1-131 of RAC, which encode the PH domain, may beexpressed as a fusion protein, advantageously withglutathione-S-transferase (GST), subsequently cleaving the fusionprotein with thrombin and isolating the domain by protein purificationtechniques, such as FPLC. This method gives a relatively small yield ofpure soluble PH domain.

Carboxy and kinase domains are likewise advantageously synthesised asfusion proteins, for instance as GST fusions.

RAC-PK or a fragment thereof for use in the present invention may beprepared as set forth in UK patent application No. 9525702.8.Alternatively, RAC-PK may be expressed in recombinant cell culture.Baculovirus vectors, specifically intended for insect cell culture, areespecially preferred and are widely obtainable commercially, e.g., fromInvitrogen and Clontech. Other virus vectors capable of infecting insectcells are known, such as Sindbis virus. See Hahn, Hahn, Braciale andRice, Proc Natl Acad Sci USA, Vol. 89, No. 7, pp. 2679-2683 (1992). Thebaculovirus vector of choice [reviewed by Miller, Ann Rev Microbiol,Vol. 42, pp. 177-199 (1988)] is Autographa californica multiple nuclearpolyhedrosis virus (AcMNPV).

Typically, the heterologous gene replaces at least in part thepolyhedrin gene of AcMNPV, since polyhedrin is not required for virusproduction. In order to insert the heterologous gene, a transfer vectoris advantageously used. Transfer vectors are prepared in E. coli hostsand the DNA insert is then transferred to AcMNPV by a process ofhomologous recombination. Baculovirus techniques useful in the presentinvention are standard and well-known in the art. See O'Reilly et al.,Baculovirus expression vectors; A laboratory manual, Oxford UniversityPress Inc., New York (1994), as well as in literature published bysuppliers of commercial baculovirus kits, e.g., Pharmingen.

Incubation conditions will vary according to the precise method used todetect the interaction between the PH domain and the screened compound.In the case of transcription activation detection systems, such as theyeast two-hybrid system, incubation conditions are suitable for genetranscription, such as those prevailing inside a living cell. Otherdetection systems, however, will require different incubationconditions. For example, if the detection of interaction is based onrelative affinity in a chromatographic assay, e.g., as is known inaffinity chromatography, conditions will be adjusted to promote bindingand then gradually altered, such that the point at which the screenedcompound no longer binds to the RAC-PK PH domain may be determined.

The detection method may employ the natural fluorescence of tryptophanat position 22 (Trp²²) in the RAC-PK PH domain, which is inhibited bycertain interactions with the domain, as set forth in UK patentapplication No. 9525703.6. Briefly, fluorescence of the amino-terminalTrp residue in the PH domains of certain PH domain containing proteinsmay be detected by exciting the molecule to fluoresce at the appropriatefrequency and monitoring the emission. The N-terminal Trp²² of RAC,e.g., fluoresces at 345 nm when excited at 290 nm. Techniques formonitoring protein fluorescence are widely-known in the art. We haveshown that the PH domain of RAC-PK binds phospholipid with highaffinity, which suggests that RAC-PK may be membrane-bound in vivo viathe PH domain. Binding of phospholipid to the RAC-PK PH domain quenchesthe natural fluorescence of the N-terminal Trp²². Interaction of the PHdomain with the cell membrane is believed to be important for the stableinteraction of RAC-PK with membrane bound partners in signallingpathways, such that disruption of this interaction will lead tomodulation of the signalling effect through the dissociation of thesignalling molecule from the cell membrane. The modulation could beeither down-regulating, e.g., if the otherwise stable interaction of themolecule with membrane-bound partners is a stimulatory interaction, orup-regulating, in the event that the interaction is an inhibitoryinteraction. Accordingly, a compound which is a candidate modulator ofsignal response may be screened for by means of a method comprising thesteps of:

-   -   (a) incubating the compound with the PH domain of a signalling        molecule which is capable of fluorescing; and    -   (b) determining the phospholipid-induced modulation in the        fluorescence of the PH domain, an alteration of the fluorescence        in the presence of the compound being indicative of a functional        interaction between the compound and the PH domain.

In this case, the incubation conditions will be adjusted to facilitatethe detection of fluorescence at 345 nm when the PH domain is excited ata frequency of 290 nm.

Incubation according to the invention may be achieved by a number ofmeans, but the basic requirement is for RAC-PK or a fragment thereof andthe screened compound to be able to come into contact with each other.This may be achieved by admixing RAC-PK or a fragment thereof and thecompound, or by producing them in situ, such as by expression of nucleicacids encoding them. Where the RAC-PK or RAC-PK fragment and/or thecompound are in the form of fusions with other polypeptides, they may beexpressed as such in situ.

Preferably, the method of the invention is based on a two-hybrid system.Such systems detect specific protein:protein interactions by exploitingtranscriptional activators having separable DNA-binding andtranscription activating domains, such as the yeast GAL4 activator. Areporter gene is operatively linked to an element responsive to thetranscriptional activator being used, and exposed to RAC-PK or afragment thereof and the compound to be screened, one of which iscomplexed to the transcription activating domain of the transcriptionalactivator and the other of which is joined to the DNA binding domainthereof. If there is a specific interaction between RAC-PK or a fragmentthereof and the compound, the DNA binding and transcription activatingdomains of the transcriptional activator will be brought intojuxtaposition and transcription from the reporter gene will beactivated.

Alternatively, the detection may be based on observed binding betweenRAC-PK or a fragment thereof, such as its PH domain or its catalyticdomain, and the screened compound, or a fragment thereof. For example,the interaction between RAC-PK and the insulin mediator GSK3 is detectedhereinbelow by monitoring the interaction of a peptide surrounding themajor phosphorylation site of GSK3 known to be responsible for itsinactivation with RAC-PK. In a similar manner, the involvement of RAC-PKon the activation or inactivation of a particular compound may bescreened for by monitoring the interaction of a portion thereof known tobe involved in modulation events with RAC.

RAC-PK or a fragment thereof may be used to screen for compounds whichbind thereto by incubating it with the compound to be screened andsubsequently “pulling down” RAC-PK complexes with a RAC-specificantibody. Antibodies suitable for immunoprecipitation or immuno-affinitychromatography may be prepared according to conventional techniques,known to those of ordinary skill in the art, and may be monoclonal orpolyclonal in nature. For example, see Lane et al., EMBO J, Vol. 11, No.5, pp. 1743-1749 (1992). After the RAC-compound complex has beenisolated by affinity, the compound may be dissociated from the RAC-PKantibody and characterised by conventional techniques.

The interaction of RAC-PK or a fragment thereof with the screenedcompound may also be observed indirectly. For example, an inhibitor oractivator of RAC-PK function may be detected by observing the effects ofRAC-PK on a substrate in the presence or absence of the compound.

The activity of RAC-PK or the catalytic domain thereof may be assessedby means of a kinase activity assay, employing a substrate for thekinase. For example, myelin basic protein (MBP) may be used, inaccordance with established assay procedures. Physiological substrates,such as GSK3, may also be used. Alternatively, RAC-PK activity may beassessed by determining the degree of activating phosphorylation ofRAC-PK itself. Advantageously, phosphorylation on residues normallyimplicated in kinase activation is assessed. RAC-PK, as disclosed in UKpatent application No. 9525702.8, is preferentially activated byphosphorylation at Ser and Thr residues.

The assay of the invention may be used to measure the direct effect ofthe candidate compound on RAC, or it may be used to determine the effectof the compound on a kinase acting upstream thereof in a signallingpathway. In the latter situation, RAC-PK acts as a substrate for theupstream kinase and the activity of the upstream kinase is assessed bydetermining the phosphporylation state or the activity of RAC-PK.

In order to obtain a meaningful result, the activity of RAC-PK exposedto the candidate immunosuppressive or antiproliferative agent should becompared to the activity of RAC-PK not exposed to the agent, amodulation of RAC-PK activity being indicative of potential as amodulator of cell proliferation and/or insulin signal transduction.

Promising compounds may then be further assessed by determining theproperties thereof directly, for instance, by means of a cellproliferation assay. Such an assay preferably involves physicaldetermination of proliferation in cells which have been subjected tokinase activation by a phosphatase inhibitor, exposed to the candidateRAC-PK modulator and optionally subsequently stimulated with a mitogen,such as a growth factor, IL-2 or PMA. More simply, the assay may involveexposure of unstimulated cells to the candidate modulator, followed bystimulation with a phosphatase inhibitor.

The invention further comprises the use of RAC-PK or a fragment thereofin a screening system. The screening system is preferably used to screenfor compounds which are modulators of insulin activity, particularlywhere that activity is related to glycogen metabolism or cellproliferation.

Kits useful for screening such compounds may be prepared, and willcomprise essentially RAC-PK or a fragment thereof together with meansfor detecting an interaction between RAC-PK and the screened compound.Preferably, therefore, the screening kit comprises one of the detectionsystems set forth hereinbefore.

RAC-PK for use in kits according to the invention may be provided in theform of a protein, e.g., in solution, suspension or lyophilised, or inthe form of a nucleic acid sequence permitting the production of RAC-PKor a fragment thereof in an expression system, optionally in situ.Preferably, the nucleic acid encoding RAC-PK or a fragment thereofencodes it in the form of a fusion protein, e.g., a GST fusion.

In a still further embodiment, the invention provides a compound whichinteracts directly or indirectly with RAC-PK or a fragment thereof. Inthe case of indirectly acting compounds, agents, such as insulin andwortmannin, are excluded. Such a compound may be inorganic or organic,e.g., an antibiotic, and is preferably a proteinaceous compound involvedin intracellular signalling. For example, the compound may be CTBP (SEQID NOs: 1 and 2).

Compounds according to the invention may be identified by screeningusing the techniques described hereinbefore, and prepared by extractionfrom natural sources according to established procedures, or bysynthesis, especially in the case of low molecular weight chemicalcompounds. Proteinaceous compounds may be prepared by expression inrecombinant expression systems, e.g., a baculovirus system as describedhereinbefore or in a bacterial system, e.g., as described in UK patentapplication No. 9525705.1. Proteinaceous compounds are mainly useful forresearch into the function of signalling pathways, although they mayhave a therapeutic application.

Low molecular weight compounds, on the other hand, are preferablyproduced by chemical synthesis according to established procedures. Theyare primarily indicated as therapeutic agents. Low molecular weightcompounds and organic compounds in general may be useful as insulinmimics or anti-proliferative agents.

The present invention further provides the use of RAC-PK, its analogues,isoforms, inhibitors, activators and/or the functional equivalentsthereof to regulate glycogen metabolism and/or protein synthesis, inparticular, in disease states where glycogen metabolism and/or proteinsynthesis exhibits abnormality, e.g., in the treatment of type IIdiabetes; also in the treatment of cancer, such as ovarian, breast andpancreatic cancer. A composition comprising such agents is also coveredby the present invention, and the use of such a composition fortreatment of disease states where glycogen metabolism and/or proteinsynthesis exhibit abnormality.

The present invention also provides a novel peptide comprising the aminoacid sequence Arg-xaa-Arg-Yaa-zaa-Ser/Thr-Hyd, where Xaa is any aminoacid, Yaa and Zaa are any amino acid (preferably not Gly), and Hyd is alarge hydrophobic residue, such as Phe or Leu, or a functionalequivalent thereof. Represented in single letter code, a suitablepeptide would be RXRX′X′S/TF/L, where X′ can be any amino acid, but ispreferably not Gly; Gly can in fact be used, but other amino acids arepreferred. Typical peptides include GRPRTSSFAEG (SEQ ID NO: 5), RPRAATC(SEQ ID NO: 6) or functional equivalents thereof. The peptide is asubstrate for measuring RAC-PK activity.

The invention also provides a method for screening for substances whichinhibit the activation of RAC-PK in vivo by preventing its interactionwith PIP3 or PI3,4-bisP.

Thus the invention also provides a method of determining the ability ofa substance to affect the activity or activation of RAC-PK, the methodcomprising exposing the substance to RAC-PK and phosphatidyl inositolpolyphosphate, i.e., PIP3 or PI3,4-bisP, etc) and determining theinteraction between RAC-PK and the phosphatidyl inositol polyphosphate.The interaction between RAC-PK and the phosphatidyl inositolpolyphosphate can conveniently be measured by assessing thephosphorylation state of RAC-PK, preferably at T308 and/or S473, e.g.,by measuring transfer of radiolabelled ³²P from the PIP3, e.g., to theRAC-PK and/or by SDS-PAGE.

The method of the invention can also be used for identifying activatorsor inhibitors of GSK3, such a method can comprise exposing the substanceto be tested to GSK3, and optionally, a source of phosphorylation, anddetermining the state of activation of GSK3, optionally by determiningthe state of its phosphorylation. This aspect of the invention can beuseful for determining the suitability of a test substance for use incombatting diabetes, cancer, or any disorder which involves irregularityof protein synthesis or glycogen metabolism.

The invention also provides a method for screening for inhibitors oractivators of enzymes that catalyse the phosphorylation of RAC-PK, themethod comprising exposing the substance to be tested to:

-   -   (a) one or more enzymes upstream of RAC-PK;    -   (b) RAC-PK; and optionally    -   (c) nucleoside triphosphate and determining whether, and        optionally to what extent the RAC-PK has been phosphorylated on        T308 and/or S473.

Also provided is a method of identifying agents able to influence theactivity of GSK3, said method comprising:

-   -   (a) exposing a test substance to a substrate of GSK3; and    -   (b) detecting whether, and optionally, to what extent said        peptide has been phosphorylated.

The test substance may be an analogue, isoform, inhibitor or activatorof RAC-PK, and the above method may be modified to identify those agentswhich stimulate or inhibit RAC-PK itself. Thus such a method maycomprise the following steps:

-   -   (a) exposing the test substance to a sample containing RAC-PK,        to form a mixture;    -   (b) exposing said mixture to a peptide comprising the amino acid        sequence defined above or a functional equivalent thereof        (usually in the presence of Mg₂+ and ATP); and    -   (c) detecting whether, and optionally, to what extent said        peptide has been phosphorylated.

In this aspect, the method of the invention can be used to determinewhether the substance being tested acts on RAC-PK or directly on GSK3.This can be done by comparing the phosphorylation states of the peptideand RAC-PK; if the phosphorylation state of GSK3 is changed but that ofRAC-PK is not then the substance being tested acts directly on GSK3without acting on RAC-PK. In a further aspect, the present inventionprovides a method of treatment of the human or non-human, preferablymammalian, animal body, said method comprising administering RAC-PK, itsanalogues, inhibitors, stimulators or functional equivalents thereof tosaid body. Said method affects the regulation of glycogen metabolism inthe treated body.

The method of treatment of the present invention may be of particularuse in the treatment of type II diabetes, where desirably an activatorof RAC-PK is used, so that the down-regulation of GSK3 activity due tothe action of RAC-PK is enhanced.

The method of treatment of the present invention may alternatively be ofparticular use in the treatment of cancer, such as ovarian cancer, wheredesirably an inhibitor of RAC-PK is used, so that the down-regulation ofGSK3 activity due to the action of RAC-PK is depressed. Other cancersassociated with irregularities in the activity of RAC-PK and/or GSK3 mayalso be treated by the method, such as pancreatic cancer and breastcancer.

Stimulation of RAC-PK with insulin increases activity 12-fold within 5minutes and induces its phosphorylation at Thr308 and Ser473. RAC-PKtransiently-transfected into cells can be activated 20-fold in responseto insulin and 46-fold in response to IGF-1 and also becamephosphorylated at Thr308 and Ser473. The activation of RAC-PK and itsphosphorylation at both Thr308 and Ser473 can be prevented by thephosphatidylinositol (P1) 3-kinase inhibitor wortmannin. Thephosphorylation of Thr308 and Ser473 act synergistically to activateRAC-PK.

MAPKAP kinase-2-phosphorylated RAC-PK at Ser473 in vitro increasesactivity 7-fold, an effect that can be mimicked (5-fold activation) bymutating Ser473 to Asp. Mutation of Thr308 to Asp also increases RAC-PKactivity 5-fold and subsequent phosphorylation of Ser473 by MAPKAPkinase-2 stimulates activity a further 5-fold, an effect mimicked(18-fold activation) by mutating both Thr308 and Ser473 to Asp. Theactivity of the Asp308/Asp473 double-mutant was similar to that of thefully phosphorylated enzyme and could not be activated further byinsulin. Mutation of Thr308 to alanine (Ala) did not prevent thephosphorylation of transfected RAC-PK at Ser473 after stimulation of 293cells with insulin or IGF-1, but abolished the activation of RAC-PK.Similarly, mutation of Ser473 to Ala did not prevent the phosphorylationof transfected RAC-PK at Thr308 but greatly reduced the activation oftransfected RAC-PK. This demonstrates that the activation of RAC-PK byinsulin or IGF-1 results from the phosphorylation of Thr308 and Ser473and that phosphorylation of both residues is preferred to generate ahigh level of RAC-PK activity in vitro or in vivo. Also, phosphorylationof Thr308 in vivo is not dependent on the phosphorylation of Ser473 orvice versa, that the phosphorylation of Thr308 and Ser473 are bothdependent on PI 3-kinase activity and suggest that neither Thr308 norSer473 phosphorylation is catalyzed by RAC-PK itself.

Thus, it is preferred that the present invention incorporates the use ofany agent which affects phosphorylation of RAC-PK at amino acids 308and/or 473, e.g. insulin, inhibitors of PI 3-kinase, such as wortmanninor the like. The use of RAC-PK, itself altered at amino acids 308 and/or473, e.g., by phosphorylation and/or mutation, is also suitable.

In a variation of the method of the present invention, stimulation orinhibition of RAC-PK may be assessed by monitoring the phosphorylationstates of amino acids 308 and/or 473 on RAC-PK itself.

Different isoforms of RAC-PK may be used or targeted in the presentinvention, e.g., RAC-PKα, β or γ.

We have observed that modulation of RAC-PK activity appears to beeffected by reversible phosphorylation, in which the equilibrium of thephosphorylation/dephosphorylation reaction is shifted in order to changethe levels of active RAC-PK with respect to its inactive form. Build-upof the active form may therefore be promoted by inhibition of thedephosphorylation reaction, achieved by treatment with a phosphataseinhibitor.

A surprising aspect of the present invention is that tyrosinephosphatase inhibitors, such as vanadate, are able to activate RAC-PKnotwithstanding the fact that, as is disclosed herein, this kinase isactivated by phosphorylation at Ser and Thr residues.

It is known that vanadate activates p70S6K. The invention accordinglydoes not extend to the use of vanadate to activate p70S6K. However, theuse of phosphatase inhibitors, such as okadaic acid, which acts througha quite different mechanism, is part of the present invention.

As referred to herein, the signalling pathways are the activationcascades which ultimately regulate signal transduction and kinases ofthese pathways are kinases whose in vivo targets include at least oneentity which contributes to such signal transduction. Preferably, thesignalling pathways of the invention are insulin-dependent signallingpathways, which are responsible for transduction of signals from insulinand other growth factors. Without in any way wishing to place anylimitation on the present invention, one such a pathway is believed tobe triggered in vivo by binding of growth factors, such as insulin andthe like to their receptors, which stimulates inter alia PI-3K. PI-3K inturn directly or indirectly phosphorylates RAC-PK, which indirectlyleads to the eventual phosphorylation of p70S6K.

Treatment of kinases according to the invention in order to activatethem requires the exposure of the kinase to a phosphorylating agent,such as another kinase of the signaling pathway and the phosphataseinhibitor. This may be accomplished, e.g., in vitro by:

-   -   (a) incubating together a kinase of a signalling pathway, an        agent capable of phosphorylating the kinase in order to activate        it, and a phosphatase inhibitor; and    -   (b) purifying the kinase from the incubation mixture.

The phosphorylating agent should be effective to phosphorylate thekinase on residues which lead to activation thereof. In the case ofRAC-PK, the phosphorylating agent advantageously targets Ser and Thrresidues.

Preferably, the phosphorylating agent is one or more kinases of thesignalling pathway which act, in the presence of suitable activatingfactors, to phosphorylate and thereby activate the kinase of interest.Preferably, this is accomplished by recovering active kinase enzyme formphosphatase-inhibitor treated cells, which contain the requiredsignaling pathway kinases.

In the context of the present invention, in vitro signifies that theexperiment is conducted outside a living organism or cell. In vivoincludes cell culture. Treatment of cells in vivo with phosphataseinhibitors is especially effective for the preparation of active RAC-PK.However, since RAC-PK and other signaling kinases, e.g., p70S6K, are onthe same pathway, activation of RAC-PK results in the activation ofother kinases on the same signalling pathway, e.g., p70S6K itself. Theinvention therefore includes a method for activating kinases onsignalling pathways in general, except for p70S6K, especially where suchkinases are downstream of RAC-PK in the pathway.

Cells which produce kinases which may be used in the present inventiongenerally include any cell line of mammalian origin, especiallyfibroblast cell lines, such as RAT-1, COS or NIH 3T3. Swiss 3T3 cellsare particularly preferred. Where human cell lines are used, humanembryonic kidney 293 cells are preferred.

Phosphatase inhibitors are agents which inhibit proteindephosphorylation by inhibiting the activity of phosphatase enzymes. Aphosphatase has essentially the inverse activity of a kinase, andremoves phosphate groups.

Examples of phosphatase inhibitors are vanadate and okadaic acid, withvanadate being the more effective agent in the case of RAC-PK. However,the action of vanadate is believed to be indirect, since it is aspecific tyrosine phosphatase inhibitor and RAC-PK does not appear to bestimulated by tyrosine phosphorylation. Okadaic acid, on the other hand,which is known to act directly on phosphatase PP2A, appears to directlyinhibit dephosphorylation of RAC-PK.

The use of other phosphatase inhibitors is envisaged and limited only bythe suitability of such inhibitors for administration to the particularcell line being used. Vanadate and okadaic acid are believed to begenerally applicable, but those of skill in the art will recognize thatother phosphatase inhibitors are available and that their activity andsuitability may easily be determined by routine empirical testing. Forexample, phosphatase inhibitors which may be suitable in the presentinvention include calyculin A, cantharidic acid, cantharidin, DTX-1,microcystin, nodularin and tautomycin. These and other phosphataseinhibitors are available commercially, e.g., from Calbiochem.

The phosphatase inhibitor is administered to cells in their normalgrowth medium, which may be serum free. Serum is itself observed tostimulate kinase activity, but is expensive and its function may besubstituted by a phosphatase inhibitor according to the presentinvention. Suitable concentrations of phosphatase inhibitors includelevels from 0.01-10 mM, preferably 0.1-1 mM. The most preferredconcentration for vanadate is 0.1 mM.

The method of the invention may comprise additional steps intended toisolate the desired active kinase from the cells in which it isproduced. Such steps are conventional procedures familiar to thoseskilled in the art and may be substituted for equivalent processeswithin the scope of the invention. The preferred process, however,comprises the steps of homogenizing the cells, removing cell debris,e.g., by centrifugation, and separating the desired kinase by affinitypurification.

Homogenization may be carried out in a standard isotonic lysis buffer,advantageously containing a proteinase inhibitor, such as phenylmethylsulphonyl fluoride (PMSF) and a phosphatase inhibitor in order toinhibit deactivation of the kinase during the purification procedure.The cells are disrupted, thereby releasing the cytoplasmic and nuclearcontents thereof into the lysis buffer.

Cell debris is then advantageously removed from the lysed cellularpreparation, preferably by centrifuging the mixture in order to pelletall particulate matter. Only the soluble fraction remains in thesupernatant.

The supernatant can then be subjected to standard protein purificationtechniques in order to isolate the kinase of interest if desired.Preferred methods, especially for relatively low volume preparations,involve affinity chromatography. Such techniques may employ ananti-kinase antibody or antiserum immobilised to a suitable matrix.Other immobilized binding agents, such as substrate analogues, may beemployed.

Antibodies useful for immunoseparation of activated kinases according tothe invention may be prepared according to techniques known in the art.In order to prepare a polyclonal serum, e.g., an antigenic portion ofthe desired kinase, consisting of a peptide derived therefrom, such as aC-terminal peptide, or even the whole kinase, optionally in the presenceof an adjuvant or conjugated to an immunostimulatory agent, such askeyhole limpet haemocyanin, is injected into a mammal, such as a mouseor a rabbit, and antibodies are recovered therefrom by affinitypurification using a solid-phase bound kinase or antigenic portionthereof. Monoclonal antibodies may be prepared according to establishedprocedures.

Alternatively, and especially for larger scale preparations, separationprocedures not involving affinity chromatography may be used.

For example, numerous methods are available in the art for separatingpolypeptides on the basis of size, such as chromatography and gelelectrophoresis. Preferred are methods which perform a purificationfunction, as well as a size separating function, while not introducingunacceptable contaminants. Thus, methods, such as step or continuousgradient centrifugation, particularly using sucrose gradients, dialysistechniques using controlled-pore membranes and membrane (Amicon)centrifugation, are preferred. Especially preferred, however, is sizeexclusion chromatography, typically performed using porous beads as thechromatographic support. Size exclusion chromatography is, e.g.,described by Stellwagen in Deutscher, Ed., Guide to ProteinPurification, Academic Press, Inc., San Diego, Calif., pp. 317-328(1990).

Alternative purification methods, described in general in Deutscher(1990), supra, include chromatography based on separation by chargedifference, such as ion exchange chromatography using an exchange group,such as DEAE or CM bound to a solid phase packing material, such ascellulose, dextran, agarose or polystyrene. Other methods includehydroxyapatite column chromatography [see, e.g., Gorbunoff, MethodsEnzymol, Vol. 117, pp. 370-380 (1985)], and general affinitychromatography using glass beads or reactive dyes as affinity agents.

Advantageously, cation exchange chromatography may be employed, suchthat protein elution can be tailored to take into account the known orestimated PI of the kinase in question. The PI for any kinase may bedetermined experimentally, by isoelectric focusing. In this manner, itis possible selectively to elute from the cation exchange resin thoseproteins having a PI at or around that of the kinase, which results in ahigh degree of purification.

The invention further provides the use of an active kinase preparedaccording to the invention in a method for screening potentialmodulators of signalling pathways. Thus, the claimed method may comprisethe additional step of exposing the kinase to a potential inhibitor andsubsequently assessing the activity of the kinase in order to determinethe effectiveness of the modulator.

The invention accordingly provides a method for screening candidatemodulators of signalling pathways comprising:

-   -   (a) incubating together a kinase of a signalling pathway and a        phosphatase inhibitor;    -   (b) adding a candidate modulator of the signalling pathway; and    -   (c) determining the activity of the kinase.

The exposure to the modulator may be performed on the activated orinactivated kinase either in a cell-free environment, optionally afterpurification of the kinase from the crude cellular preparation, or insitu in the cells which produce the kinase, after phosphatase inhibitoractivation. Steps (a) and (b) may therefore be reversed, or conductedcontemporaneously.

In step (a), especially if the assay is to be performed in vitro, anagent capable of phosphorylating the kinase may be added to theincubation mixture. Phosphatase inhibitors activate kinases bypreventing dephosphorylation, so a phosphorylating agent will berequired. Advantageously, the phosphorylating agent is a kinase of aninsulin-dependent signalling pathway or an analogue thereof. Moreover,factors may be required to initiate or assist signal transduction in thesignalling pathway. For example, it the compound being tested is arapamycin analogue which binds FKBP, FKBP will be required in theincubation mixture.

Preferably, however, the procedure is carried out in vivo in cellscontaining kinases of the signalling pathway. In such an assay, thephosphatase inhibitor replaces serum or other agents previously employedas external stimulating agents to activate kinases of the signallingpathway.

The activity of the kinase may be assessed by means of a kinase activityassay, employing a substrate for the kinase. For example, MBP may beused, in accordance with established assay procedures. Physiologicalsubstrates, such as the 40S ribosomal subunit, or S6, may also be used.Alternatively, kinase activity may be assessed by determining the degreeof phosphorylation of the kinase. Advantageously, phosphorylation onresidues normally implicated in kinase activation is assessed. Theidentification of such residues, which is part of the present invention,is set forth below.

The assay of the invention may be used to measure the direct effect ofthe candidate compound on the assayed kinase, or it may be used todetermine the effect of the compound on a kinase acting upstream thereofin the signalling pathway. In the latter situation, the assayed kinaseacts as a substrate for the upstream kinase and the activity of theupstream kinase is assessed by determining the phosphporylation state orthe activity of the assayed kinase.

In order to obtain a meaningful result, the activity of the assayedkinase exposed to the candidate modulator of the signalling pathwayshould be compared to the activity of the kinase not exposed to theagent, an inhibition of kinase activity being indicative of potential asan immunosuppressive or anti-proliferative.

Compounds which demonstrate elevated levels of kinase inhibition maythen be further assessed by determining the immunosuppressive oranti-proliferative properties thereof directly, for instance, by meansof a cell proliferation inhibition assay. Such an assay preferablyinvolves physical determination of T-cell proliferation in cells whichhave been subjected to kinase activation by a phosphatase inhibitor,exposed to the candidate kinase inhibitor and optionally subsequentlystimulated with a mitogen, such as a growth factor, IL-2 or PMA. Moresimply, the assay may involve exposure of unstimulated cells to thecandidate inhibitor, followed by stimulation with a phosphataseinhibitor.

According to a further aspect of the invention, we have been able todetermine which sites are important for the phosphorylation of kinases,particularly those of the p70^(S6K)/RAC-PK family. Surprisingly, themajority of activating phosphorylation appears to take place on Ser andThr residues. It is known that phosphorylation may in certain cases bemimicked by replacement of the phosphorylated amino with an acidic aminoacid, such as aspartic acid or glutamic acid.

The invention accordingly provides a recombinant RAC-PK protein whereinat least one threonine residue involved in activation of the kinasethrough phosphorylation in vivo is replaced with an acidic amino acidresidue. Moreover, the invention provides a method for screeningcompounds which inhibit signalling by RAC-PK comprising exposing cellstreated with the constitutively active recombinant RAC-PK to thecompounds.

For example, an important activating residue is Thr308, present in theso-called T-loop between subdomains 7 and 8 of the kinase. A generalguide to kinase structure is given in Woodgett (1994), supra.Substitution of Thr308 with aspartic acid results in a clear increase inbasal activity of the kinase, which however retains a potential forfurther activation. The invention therefore provides a RAC-PK in whichThr308 has been mutated to Asp.

Preferably, Ser473 is additionally mutated to Asp. Phosphorylation ofthis residue is required for full-activation of RAC-PK in vivo, and theThr308/Ser473 double-mutant (both residues converted to Asp) shows aconstitutive activity 18-fold higher than native RAC-PK. Thedouble-mutant does not retain the capability for further activation.

Constitutively active kinases according to the invention may be employedin place of the phosphatase inhibitor activated kinase in screeningtechniques as described herein. Advantageously, such constitutivelyactivated kinases require no external stimulating agents.

The present invention will now be described in more detail in theaccompanying examples which are provided by way of non-limitingillustration, and with reference to the accompanying drawings.

EXAMPLE 1

Specific Interaction of RAC-PK with IMPDH

a. Bacterial and Yeast Strains

All yeast strains and plasmids for two-hybrid experiments are obtainedfrom Clontech (Palo Alto, Calif.) as components of the MATCHMAKER TwoHybrid System or from Dr. Nathans, Howard Hughes Medical Institute,Baltimore, Md. Yeast strains SFY526, e.g., MATa, Ura3-52, His3-200,Ade2-101, Lys2-801, Trp1-901, Leu2-3, 112, can^(r), Gal4-542, Gal80-538and Ura3::GAL1-lacZ; HF7c, e.g., MATa, Ura3-52, His3-200, Lys2-801,Ade2-101, Trp1-901, Leu2-3, 112, Gal4-542, Gal80-538, Lys2::Gal1-His3and Ura3::(Gal4 17-mer)₃-CYC1 -LacZ; and PCY2 [see Chevray and Nathans,Proc Natl Acad Sci USA, Vol. 89, No. 13, pp. 5789-5793 (1992)], e.g.,MATα, His3-200, Ade2-101, Lys2-801, Trp1-63, Leu2-3, Gal4-542, Gal80-538and Ura3::Gal1-LacZ, are used to assay for protein-protein interactions.Yeast strain HF7c is used for library screening. SFY526 and PCY2 havethe upstream activating sequence and TATA sequence of the GAL1 promoterfused to the LacZ gene. In HF7c, His3 is fused to a Gal1 promotersequence and LacZ is fused to three copies of a 17-mer Gal4 consensussequence plus the TATA sequence of the CYC1 promoter. Both His3 and LacZare responsive to Gal4 activation. Yeast techniques includingtransformation are performed according to the instructions in theMATCHMAKER Two Hybrid System and as described Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, New York, N.Y.(1994). The bacterial strains XL1 -blue (Statagene) and JM109 areemployed in the cloning of plasmids and the production of GST fusionproteins. The bacterial strains JM109(DE3), BL21(DE3)pLysS andBL21(DE3)pLysE (Invitrogen) are used for the production of (His)₆-taggedproteins. General molecular biological techniques are performed aspreviously described in Sambrook et al. (1989), supra; and Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing Co., NewYork, N.Y. (1986).

b. Plasmid Construction

Yeast vector plasmids containing the Gal4 DNA binding domain (aminoacids 1-147, pGBT9) and the Gal4 activation domain (amino acids 768-881,pGAD424), as well as the control plasmids pCL1 (full-length Gal4 gene),pVA3 (p53 gene), pTD1 (SV40 large T antigen), and pLAM5′ (human lamin Cgene) are from Clontech. The yeast vector pPC62, containing the Gal4 DNAbinding domain, is from Dr. Nathans. The GST fusion vector pGEX-2T isfrom Pharmacia. The baculovirus transfer vector (pVL1392) and the(His)₆-tag vector (pRSET-A) are from Invitrogen. pGBT-PH127, pGBT-PH150,pGBT-PHI-III and pGBT-PHIII-VI contain in-frame fusions of amino acids1-127, 1-150, 1-47 and 47-127 of the human RACα PH domain, respectively,with the Gal4 DNA binding domain. They are constructed by subcloning PCRfragments generated with specific oligonucleotides into the EcoRI-BamHIsites of pGBT9. pGEX-PH131, pGEX-PH-KIN, pGEX-PH-KIN-CT, pGEX-KIN-CT,pGEX-KIN and pGEX-CT contained in-frame fusions of amino acids 1-131,1-411, 1-480, 147-480, 147-411 and 411-480 of human RACα, respectively,with GST. They are constructed by subcloning PCR fragments generatedwith specific oligonucleotides into the BamHI-EcoRI sites of pGEX-2T,pGBT-PH-KIN, pGBT-PH-KIN-CT, pGBT-KIN-CT, pGBT-KIN and pGBT-CT containin-frame fusions of amino acids 1-411, 1-480, 147-480, 147-411 and411-480 of human RACα, respectively, with the Gal4 DNA binding domain.They are constructed by subcloning the appropriate BamHI-EcoRI fragmentsfrom the corresponding pGEX constructs into the PstI-XbaI sites of pPC62using PstI-BamHI and EcoRI-XbaI adapters. The XhoI-XbaI fragments fromthe resultant pPC62 plasmids are then isolated and subcloned into theXhoI-EcoRI sites of pGBT9 using a XbaI-EcoRI adapter. pGAD-IMPDH1-481,pGAD-IMPDH1-427, pGAD-IMPDH1-325, pGAD-IMPDH28-514, pGAD-IMPDH70-514,pGAD-IMPDH140 and pGAD-IMPDH428-514 contain in-frame fusions of aminoacids 1-481, 1-427, 1-325, 28-514, 70-514, 1-40 and 428-514 of humanIMPDH type II, respectively, with the Gal4 activation domain. They areconstructed by subcloning PCR fragments generated with specificoligonucleotides into the BamHI-SalI sites of pGAD424, pGEX-IMPDHcontains an in-frame fusion of the complete human IMPDH type II withGST. It is constructed by subcloning the SmaI-XhoI IMPDH fragment frompGADGH-IMPDH into the SmaI site of pGEX-2T using a XhoI-SmaI adapter.pVL1392-hRACα contained the EcoRI fragment from WI38xRAC71 [see Jones etal. (1991), supra] encompassing the full-coding region of human RACα.pRSET-PHQKKK contains an in-frame fusion of amino acids 1-116 of humanRACα with an amino-terminal (His)₆-tag and the addition of 3 lysines atthe carboxy terminus. It is constructed by subcloning an NdeI-PflMIfragment from pRK-RAC [see Jones et al. (1991), supra] into theBamHI-EcoRI sites of pRSET-A using BamHI-NdeI and PflMI-EcoRI adapters.All plasmid constructions are confirmed by restriction fragment analysisand sequencing.

c. Library Screening

To determine if RAC's PH domain could interact with other proteins wefuse it to the Gal4 DNA binding domain and screen a HeLa complementaryDNA (cDNA) library fused to the Gal4 transcriptional activation domainin the yeast reporter strain HF7c. The human HeLa S3 MATCHMAKER cDNAlibrary is purchased from Clontech. pGBT-PH127 is transformed into HF7cwith and without the control plasmids (pGAD424, pCL1 and pTD1). Coloniesfrom this transformation are tested for His3 and LacZ expression toconfirm that the PH domain alone does not activate transcription. TheHF7c transformant containing just pGBT-PH127 is then transformed withenough of the HeLa S3 cDNA library inserted into the 2-hybrid activationvector pGADGH to produce 1.0×10⁶ yeast Leu⁺/Trp⁺ transformants.Doubly-transformed cells are plated onto Leu⁻, Trp⁻ and His⁻ plates andincubated at 30° C. for 3-8 days. Positive colonies are picked,re-streaked onto triple minus plates and assayed for LacZ activity bythe filter assay. Library clones that are His⁺ and LacZ⁺ are then curedof the pGBT-PH127 plasmid and tested again for His auxotrophy and LacZactivity. Cured clones that are negative in both assays are then matedto transformants of PCY2 containing either pGBT9, pGBT-PH127, pLAM5′ orpTD1. The activation clones corresponding to the diploids which becomepositive for both His auxotrophy and LacZ activity only in the presenceof pGBT-PH127 are chosen for sequencing.

In our screen of 1.0×10⁶ primary transformants we identify 37 cloneswhich show specific interaction with RAC's PH domain, by activation ofthe reporters for His auxotrophy and LacZ activity. These clones couldbe subdivided into 6 different cDNA classes, based on the size of thecDNA insert. Upon sequencing all clones were found to encode human IMPDHtype II inclusive of the initiator methionine through to the terminationcodon of the previously cloned cDNA. See Collart and Huberman (1988),supra.

The interaction requires a complete PH domain as constructs containingeither subdomains I-III (amino acids 1-47) or subdomains IV-VI (aminoacids 47-127) alone do not show any interaction with IMPDH. The lack ofinteraction with subdomains IV-VI is significant as this region haspreviously been shown to interact weakly with the βγ-subunits ofheterotrimeric G-proteins. This interaction of IMPDH and RAC's PH domainis however inhibited in the 2-hybrid system with constructs containingRAC's kinase domain juxtaposed to RAC's PH domain as occurs in itsnatural context. This could be due to an intramolecular interaction ofRAC's PH domain with itself or another region of RAC. However, inclusionof amino acids between the PH and kinase domains (including the firstfour amino acids of the kinase domain) didn't inhibit the interaction.We also fuse RAC's PH domain to the Gal4 activation domain to test if itcould interact with any of the human RACα Gal4 DNA binding constructs.We can detect no such interaction, indicating that the PH domain doesnot self associate or form a complex with other regions of the RAC-PKmolecule in this system. The inhibition of interaction observed abovewould thus appear to be due to steric hindrance.

We construct nested amino and carboxyl-terminal deletions of IMPDH todetermine the region of the molecule responsible for interaction withRAC's PH domain. This indicates that an almost intact IMPDH molecule isrequired for the interaction. The amino-terminal boundary of the PHinteraction domain is found to lie between amino acids 28 and 70, whilethe carboxyl-terminal boundary lies between amino acids 427 and 481.

d. In Vitro Binding Studies

To test if IMPDH could interact directly with RAC's PH domain we employan in vitro binding assay system using GST fusions. GST fusions producedfrom the plasmids pGEX-2T, pGEX-PH131 and pGEX-IMPDH are expressed in E.coli XL-1 blue cells by induction with 0.1 mM IPTG for 2 hours at 24° C.The fusion proteins are purified as described [see Smith and Johnson,Gene, Vol. 67, No. 1, pp. 31-40 (1988)] except that the cells are lysedin a French Press. The human RACα PH domain (His)₆ tagged fusionproduced by B121(DE3)pLysS cells transformed with pRSET-PHQKKK isexpressed and purified as described in UK patent application No.9525705.1. Briefly, cells are induced with 0.2 mM IPTG for 2 hours at24° C. before harvesting. Cell pellets are lysed in a French Press andthe soluble PH domain is purified sequentially on Ni(II) affinity,cation exchange and gel filtration columns. Binding studies areperformed using GST fusions (2.5 μg) coupled to glutathione-agarosebeads in binding buffer (20 mM phosphate buffer, pH 7.2, 150 mM NaCl, 1%Triton X-100, 5 mM DTT) containing 2.5 μg of (His)₆-tagged PH domain orbaculovirus produced human RACα in a total volume of 100 μL. The samplesare incubated at 4° C. for 1-2 hours with agitation every 5 minutes. Thebeads are then washed 3× with buffer (20 mM phosphate buffer, pH 7.2,150 mM NaCl) before being analyzed by SDS-PAGE and stained withcoomassie blue R-250 [for (His)₆-tagged PH domain binding] or SDS-PAGEfollowed by Western blot analysis using a human RACα specific antiserum(for human RACα binding). See Jones et al., Jakubowicz and Hemmings(1991), supra. The secondary antibody is a horseradish peroxidasecoupled anti-rabbit antibody (Amersham) which is detected using the ECLmethod (Amersham) by autoradiography. In this assay we see that the(His)₆-tagged PH domain can bind to the GST-IMPDH fusion but not to GSTalone.

We also employ this assay system to test if full-length baculoviruspurified human RACα could directly interact with IMPDH. Full-lengthhuman RACα is expressed and purified from the baculovirus system asdescribed in UK patent application No. 9525702.8. Briefly, a baculovirusis constructed by co-transfection of Sf9 cells with pVL1 392-hRACα andwild-type (WT) baculovirus AcMNPV DNA and purified by limiting dilutionand detected by dot-blot hybridization. The purified virus is used toproduced human RACα in Sf9 cells. The human RACα is purified bysequential anion exchange, phospho-cellulose and gel filtrationchromatography. Here we see specific interaction of the full-lengthRAC-PK molecule with GST-IMPDH and not GST alone or the GST-PH fusion.

e. In Vivo Pull-Down Assay

Using GST-IMPDH in a pull-down assay with MCF-7 cell extracts we see aspecific association of human RACα with the GST-IMPDH and not with GST.MCF-7 cells are lysed in buffer (50 mM Tris-HCl, pH 8.0,120 mM NaCl, 1%NP-40, 1 mM EDTA, 1 mM EGTA, 30 mM pNPP, 25 mM β-glycerol phosphate, 15mM PPi, 25 mM NaF, 1 mM vanadate, 20 μM PAO, 1 mM benzamidine, 0.1 mMPMSF) using 12 strokes of a dounce homogenizer. Soluble protein from thesupernatant of lysates centrifuged at 14,000×g for 15 minutes at 4° C.are added to GST, GST-PH or GST-IMPDH protein (5 μg) attached toglutathione beads and incubated at 4° C. for 2 hours with continuousagitation. The beads are then washed 4× with lysis buffer before beinganalyzed by Western blotting as described above with the humanRACα-specific antiserum. See Jones, Jakubowicz and Hemmings (1991),supra] or an IMPDH-specific antiserum. See Collart and Huberman (1988),supra. We could also perform the converse experiment, pulling down IMPDHfrom cell lysates using the GST-PH domain fusion protein. Thus we haveshown the existence of an association between human RACα and human IMPDHtype II in 3 heterologous systems.

f. Enzyme Assays

We then assay the effect of this interaction on the activity of IMPDH.The addition of soluble PH domain as a GST fusion produced an activationof IMPDH compared to the addition of GST alone. Assays for IMPDHactivity are performed essentially [see Antonio and Wu, Biochem, Vol.33, pp. 1753-1759 (1994)], monitoring the production of XMP byabsorbance at 286 nm. The IMPDH is produced as a GST fusion purified onglutathione beads and then eluted as soluble protein with reducedglutathione. IMPDH activity is tested in the presence of either solubleGST (produced from pGEX-2T) or PH domain (produced from pGEX-PH131) at amolar ratio of IMPDH to GST/PH domain of 1:5. RAC kinase assays with thebaculovirus produced human RACα using various substrates, e.g., myelinbasic protein, GST or GST-IMPDH, are performed essentially as described.See Jones, Jakubowicz and Hemmings (1991), supra. When IMPDH is testedin RAC-PK assays using the baculovirus produced human RACα to see if itis a substrate we could detect no significant phosphorylation of theIMPDH.

EXAMPLE 2

Inhibition of GSK3

Two major kinases known to be involved in regulating theinsulin-dependent signalling pathways are MAPKAP kinase-1 and p70^(S6K).These kinases are respectively inhibited by PD98059 and rapamycin. Bothof these agents fail to inhibit phosphorylation of GSK3, suggesting thatthe kinase responsible for GSK3 inactivation is not MAPKAP kinase-1 orp70^(S6K).

L6 myotubes are incubated with both compounds and the stimulated withinsulin, as follows. Monolayers of L6 cells are grown in 6 cm petridishes to the stage of myotubes, deprived of serum overnight and thenincubated for 1 hour in 20 mM Hepes/NaOH, pH 7.4, 0.14 M NaCl, 5 mM KCl,2.5 mM MgSO₄, 1 mM CaCl₂, 25 mM glucose (HBS buffer), in the presence orabsence of 50 μM PD98059 or 100 μM LY294002. Two (2) mM 8-Br-cAMP or 0.1μM rapamycin, when added, are included for the last 15 minutes. Thecells are stimulated for 5 minutes with 0.1 μM insulin, or for timecourses of up to 10 minutes. The medium is removed by aspiration, thecells placed on ice and lysed in 0.2 mL of ice-cold buffer A, 50 mMTris-HCl, pH 7.5, 20° C., 1 mM EDTA, 1 mM EGTA, 1% (^(w)/_(v)) TritonX-100, 1 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mMsodium fluoride, 5 mM sodium pyrophosphate, 2 μM microcystin, 0.1%(^(v)/_(v)) 2-mercaptoethanol, leupeptin 4 μg/mL, 1 mM benzamidine, 1 mMphenylmethane sulphonyl fluoride, 30 μg/mL aprotinin, 30 μg/mL antipain,10 μg/mL pepstatin.

Precipitation of p42^(MAPK), MAPKAP kinase-1 or GSK3 from the celllysates by immunoprecipitation and analysis of their activity withspecific protein or peptide substrates [see Cross et al. (1994), supra]shows that insulin inactivation of GSK3 is not affected by agents (2 mM8-Br-cAMP or 0.1 μM rapamycin) which inhibit classical MAP kinase orp70^(S6K) signalling pathways.

In order to identify the kinase which inhibits GSK3 in the presence ofrapamycin and PD98059, cells are lysed as above and the cell lysates(0.3 mg) chromatographed on a 5×0.16 cm column of Mono Q [see Sutherlandand Cohen, FEBS Lett, Vol. 338, No.1, pp. 37-42 (1994)] except that thebuffer additionally contains 1 mM EGTA, 0.1 mM sodium orthovanadate and0.5% (^(w)/_(v)) Triton X-100.

The fractions (0.05 mL) assayed with the synthetic peptide GRPRTSSFAEGSEQ ID NO: 5, which corresponds to the sequence of GSK3 surrounding theserine (bold type) whose phosphorylation triggers the inactivation ofGSK 3α (Ser21) and GSK3β (Ser9). Three peaks of activity which result inphosphorylation of this peptide are eluted. These peaks are absent ifinsulin stimulation is not given, or if cells are incubated with 0.1 μMwortmannin prior to insulin stimulation. The inactivating effect ofinsulin on GSK3 is known to be prevented by administering thisconcentration of wortmannin, or 100 μM LY294002, astructurally-unrelated inhibitor of PI-3K.

All 3 peaks of phosphorylating activity can be immunoprecipitated withan anti-RAC antibody using a polyclonal rabbit antiserum directedagainst the peptide FPQFSYSASSTA SEQ ID NO: 7 raised by injectingrabbits subcutaneously with the peptide and purified by precipitationusing 50% (NH₄)₂SO₄ followed by affinity chromatography on RAC-peptidecoupled Affigel® 10 column (Bio-Rad).

In contrast, immunoprecipitating with an anti-MAPKAP kinase-1 antibodyfails to deplete any peptide phosphorylating activity from the Mono Qcolumn.

In order to determine that complete GSK3 can be inactivated by RAC,GSK3α and GSK3β are partially purified from rabbit skeletal muscle [seeSutherland and Cohen (1994), supra] and assayed with a specific peptidesubstrate. See Cross et al. (1994), supra. Each GSK3 isoform is dilutedto 15 U/mL and GSK3 activity measured after incubation for 20 minuteswith MgATP in the presence or absence of RAC. The incubation is the made20 mM in EDTA to stop the kinase reaction, incubated for 20 minutes with5 mU/mL PP2A, to reactivate GSK3, made 2 μM in okadaic acid toinactivate PP2A, and then assayed for GSK3 activity.

In the absence of RAC, GSK3 is stably active throughout the experiment.Otherwise, however, RAC-PK successfully inhibited GSK3 activity and,thisinactivation was sensitive to PP2A₁, which restored GSK3 activity. Theabsence of insulin stimulation, the presence of wortmannin or thedisruption of RAC-PK immunoprecipitation by incubation of the anti-RACantibody with the peptide immunogen all result in a lack of GSK3inactivation in this experiment.

EXAMPLE 3

To determine if RAC-PK's domain with its carboxyl-terminal extensioncould interact with other proteins we fused it to the Gal4 DNA bindingdomain and screened a HeLa cDNA library fused to the Gal4transcriptional activation domain in the yeast reporter strain HF7cfollowing the procedure of Example 1. Briefly, amino acids 147-480 ofRACα are fused in frame to GST in the expression vector pGEX-2T (seeExample 1). Appropriate BamHI-EcoRI fragment is then subcloned into thePstI-XbaI sites of yeast vector pPC62 (Dr. Nathans) using PstI-BamHI andEcoRI-XbaI linkers in order to create a Gal4 DNA binding domain-RAC-PKand C-terminal domain fusion. XhoI-XbaI fragments are then subclonedinto pGBT9 (Clontech). The HeLa S3 MATCHMAKER cDNA library is used asbefore.

In our screen of 1.5×10⁶ primary transformants, we identify 7 cloneswhich show specific interaction with RAC-PK's domain plus thecarboxyl-terminal extension, by activation of the reporters for Hisauxotrophy and LacZ activity. A detailed analysis of the specific regionof RAC-PK that the clone interacts with shows that the carboxyl-terminal69 amino acids are all that is required to confer the interaction. Wethus denote this molecule carboxyl-terminal binding protein (CTBP). Noneof the clones shows an interaction with the kinase domain alone. Thisinteraction is seen in all constructs containing the carboxyl-terminalextension including a full-length RAC-PK construct, indicating that theinteraction is not inhibited by another region of the RAC-PK molecule.Interestingly, the C-terminal domain of RAC-PK is phosphorylated inresponse to insulin activation, suggesting a role for CTBP as amodulator of insulin action.

All 7 specific interacting clones contain identical cDNA inserts of 1.3kb in length with an ALU repeat of ˜300 nt at the 3′ end (SEQ ID NO: 1).Searches of the Gene-EMBL nucleotide database using the cDNA sequencewithout the ALU repeat with the FASTA programme (GCG Package) identifyno significant homologies. The deduced amino acid sequence of the CTBPcDNA produces a short 47-residue polypeptide rich in alanines (21%) andarginines (21%). Searches of the PIR, Swiss-Prot and GP proteindatabases using FASTA (GCG Package) and the Gene-EMBL nucleotidedatabase using TFASTA (GCG Package) reveal no significant homologies tothe CTBP protein sequence.

The sequence of CTBP identified is believed to represent the 3′ end ofthe complete CTBP molecule.

To test if the novel protein CTBP could interact directly with RAC-PK weemploy an in vitro binding assay system using GST fusions, as describedin Example 1. In this assay we see specific interaction of thefull-length RAC, produced in the baculovirus system with GST-CTBP andnot GST alone.

To test if this interaction occurs in vivo we employ a pull-down assayusing the GST-CTBP fusion protein and MCF-7 cell extracts, as describedin Example 1. Here we see the specific association of the MCF-7 RACαwith the GST-CTBP but not GST alone.

EXAMPLE 4

RAC-PK Influences GSK3 Activity

FIG. 1 (a)—L6 myotubes were incubated for 15 minutes with 2 mm8-bromocyclic-AMP (8Br-cAMP) and then with 0.1 μM insulin (5 minutes).Both GSK3 isoforms were co-immunoprecipitated from the lysates andassayed before (black bars) and after (white bars) reactivation withPP2A. See Cross et al. (1994), supra. The results are presented relativeto the activity in unstimulated cells, which was 0.08±0.006 U mg⁻¹(n=10).

(b and c)—The inhibition of GSK3 by insulin (0.1 μM) is unaffected byrapamycin (0.1 μM) and PD 98059 (50 μM), but prevented by LY 294002 (100μM).

(b)—L6 myotubes were stimulated with insulin for the times indicatedwith (filled triangle) or without (filled circles) a 15 minutespre-incubation with LY 294002, and GSK3 measured as in (a). The opencircles show experiments from insulin-stimulated cells where GSK3 wasassayed after reactivation with PP2A. See Cross et al. (1994), supra.

(c)—Cells were incubated with rapamycin (triangles) or rapamycin plus PD98059 (circles) before stimulation with insulin, and GSK3 activitymeasured as in (a), before (filled symbols) and after (open symbols)pretreatment with PP2A.

(d and e)—L6 myotubes were incubated with 8Br-cAMP (15 minutes), PD98059 (60 minutes) or LY 294002 (15 minutes) and then with insulin (5minutes) as in (a-c). Each enzyme was assayed after immunoprecipitationfrom lysates, and the results are presented relative to the activitiesobtained. In the presence of insulin and absence of 8Br-cAMP, which were0.04±0.005 U mg⁻¹ (p42 MAP kinase, n=6) and 0.071±0.004 U mg⁻¹ (MAPKAPKinase⁻¹, n=6).

All the results (±s.e.m.) are for at least 3 experiments.

Monolayers of L6 cells were cultured, stimulated and lysed as describedpreviously. See Cross et al. (1994), supra. p42 MAP kinase, MAPKAPkinase 1 or GSK3-α plus GSK3-β were then immunoprecipitated from thelysates and assayed with specific protein or peptide substrates asdescribed previously. See Cross et al. (1994), supra. One unit of PKactivity was that amount which catalyzed the phosphorylation of 1 nmolof substrate in 1 minute. Where indicated, GSK3 in immunoprecipitateswas reactivated with PP2A. See Cross et al. (1994), supra.

FIG. 2—Identification of RAC-PK as the insulin-stimulated,wortmannin-sensitive and PD 98059/rapamycin-insensitive Crosstide kinasein L6 myotubes.

(a)—Cells were incubated with 50 μM PD 98059 (for 1 hour) and 0.1 μMrapamycin (10 minutes), then stimulated with 0.1 μM insulin (5 minutes)and lysed. See Cross et al. (1994), supra. The lysates (0.3 mg protein)were chromatographed on Mono Q (5×0.16 cm) and fractions (0.05 mL) wereassayed for Crosstide kinase (filled circles). In separate experimentsinsulin was omitted (open circles) or wortmannin (0.1 μM) added 10minutes before the insulin (filled triangles). The broken line shows theNaCl gradient.

Similar results were obtained in 6 experiments.

(b)—Pooled fractions (10 μL), 31-34 (lane 1), 35-38 (lane 2), 39-42(lane 3), 43-45 (lane 4), 46-49 (lane 5) and 50-53 (lane 6), from a wereelectrophoresed on a 10% SDS/polyacrylamide gel and immunoblotted withthe C-terminal anti-RAC-PKα antibody. Marker proteins are indicated. Noimmunoreactive species were present in fractions 1-30 or 54-80.

(c)—L6 myotubes were stimulated with 0.1 μM insulin and RAC-PKimmunoprecipitated from the lysates (50 μg protein) essentially asdescribed previously [see Lazar et al., J Biol Chem, Vol. 270, No. 35,pp. 20801-20807 (1995)], using the anti-PH domain antibody and assayedfor Crosstide kinase (open circles). In control experiments, myotubeswere incubated with 0.1 μM rapamycin plus 50 μM PD 98059 (opentriangles) or 2 mM 8Br-cAMP (open squares), or 0.1 μM wortmannin (filledcircles) or 100 μM LY 294002 (filled triangles) before stimulation withinsulin.

(d)—As (c) except that MAPKAP kinase-1 was immunoprecipitated from thelysates and assayed with S6 peptide (filled circles). In controlexperiments, cells were incubated with 0.1 μM rapamycin plus 50 μM PD98059 (filled triangles) or with 2 μM 8BR-cAMP (open circles) beforestimulation with insulin. In (c) and (d), the error bars denotetriplicate determinations, and similar results were obtained in 3separate experiments.

Mono Q chromatography was performed as described [see Burgering andCoffer (1995), supra], except that the buffer also contained 1 mM EGTA,0.1 mM sodium orthovanadate and 0.5% (^(w)/_(v)) Triton X-100. TwoRAC-PKα antibodies were raised in rabbits against the C-terminal peptideFPQFSYSASSTA (SEQ ID NO: 7) and bacterially-expressed PH domain ofRAC-PKα. The C-terminal antibody was affinity purified. See Jones et al.(1991), supra. The activity of RAC-PK towards Crosstide is threefoldhigher than its activity towards histone H2B and 11-fold higher than itsactivity towards myelin basic protein, the substrates used previously toassay RAC-PK. Other experimental details and units of protein kinaseactivity are given in FIG. 1.

FIG. 3—GSK3 is inactivated by RAC-PK from insulin-stimulated L6myotubes.

(a)—Cells were stimulated for 5 minutes with 0.1 μM insulin, and RAC-PKimmunoprecipitated from 100 μg of cell lysate and used to inactivateGSK3 isoforms essentially as described previously. See Sutherland,Leighton and Cohen, Biochem J, Vol. 296, Pt. 1, pp. 15-19 (1993); andSutherland and Cohen (1994), supra. The black bars show GSK3 activitymeasured after incubation with MgATP and RAC-PK as a percentage of theactivity obtained in control incubations where RAC-PK was omitted. Inthe absence of RAC-PK, GSK3 activity was stable throughout theexperiment. The white bars show the activity obtained after reactivationof GSK3 with PP2A. See Embi, Rylatt and Cohen, Eur J Biochem, Vol. 107,No. 2, pp. 519-527 (1980). No inactivation of GSK3 occurred if insulinwas omitted, or if wortmannin (0.1 μM ) was added 10 minutes before theinsulin, or if the anti-RAC-PK antibody was incubated with peptideimmunogen (0.5 mM) before immunoprecipitation. The results (±s.e.m.) arefor 3 experiments (each carried out in triplicate).

(b)—Inactivation of GSK3-β by HA-RAC-PKα. cDNA encoding HA-RAC-PKα wastransfected into COS-1 cells, and after stimulation for 15 minutes with0.1 mM sodium pervanadate the tagged protein kinase wasimmunoprecipitated from 0.3 mg of lysate and incubated for 20 minuteswith GSK3-β and MgATP. In control experiments, pervanadate was omitted,or WT RAC-PKα replaced by vector (mock translation) or by akinase-inactive mutant of RAC-PKα in which Lys179 was mutated to Ala(K179A). Similar results were obtained in 3 separate experiments. Thelevels of WT and K179A-RAC-PKα in each immunoprecipitate were similar ineach transfection.

In a GSK3-α and GSK3-β were partially-purified, assayed, inactivated byRAC-PK and reactivated by PP2A from rabbit skeletal muscle as describedpreviously. See Sutherland, Leighton and Cohen, Biochem J (1993), supra;and Sutherland and Cohen (1994), supra. There was no reactivation incontrol experiments in which okadaic acid (2 μM) was added before PP2A.

FIG. 4—Identification of the residues in GSK3 phosphorylated by RAC-PKin vitro and in response to insulin in L6 myotubes.

(a)—GSK3-β was maximally inactivated by incubation with RAC-PK andMg-[γ-³²P]ATP and after SDS-PAGE, the ³²P-labelled GSK3-β (M_(r) 47K)was digested with trypsin¹¹ and chromatographed on a C18-column. SeeSutherland, Leighton and Cohen, Biochem J (1993), supra. Fractions (0.8mL) were analyzed for ³²P-radioactivity (open circles), and the diagonalline shows the acetonitrile gradient.

(b)—The major phosphopeptide from a (400 c.p.m.) was subjected tosolid-phase sequencing [see Sutherland, Leighton and Cohen, Biochem J(1993), supra], and ³²P-radioactivity released after each cycle of Edmandegradation is shown.

(c)—GSK3-α and GSK3-β were co-immunoprecipitated from the lysates of³²P-labelled cells, denatured in SDS, subjected to SDS-PAGE, transferredto nitrocellulose and autoradiographed. See Saito, Vandenheede andCohen, Biochem J (1994). Lanes 1-3, GSK3 isoforms immunoprecipitatedfrom unstimulated cells; lanes 4-6, GSK3 isoforms immunoprecipitatedfrom insulin-stimulated cells.

(d)—GSK3 isoforms from (c) were digested with trypsin, and the resultingphosphopeptides separated by isoelectric focusing [see Saito,Vandenheede and Cohen (1994), supra] and identified by auto-radiography.Lanes 1 and 4 show the major phosphopeptide resulting from in vitrophosphorylation of GSK3-β by RAC-PK and MAPKAP kinase-1, respectively;lanes 2 and 5, the phosphopeptides obtained from GSK3-β and GSK3-α,immunoprecipitated from unstimulated cells; lanes 3 and 6, thephosphopeptides obtained from GSK3-β and GSK3-α immunoprecipitated fromcells stimulated for 5 minutes with 0.1 μM insulin; the arrow denotesthe peptides whose phosphorylation is increased by insulin. The PIvalues of two markers, Patent Blue (2.4) and azurin (5.7) are indicated.

In (a), RAC-PKα was immunoprecipitated with the C-terminal antibody fromthe lysates (0.5 mg protein) of insulin-stimulated L6 myotubes and usedto phosphorylate GSK-β. In (c), three 10 cm diameter dishes of L6myotubes were incubated for 4 hours in HEPES-buffered saline [see Crosset al. (1994), supra] containing 50 μM PD 98059, 100 nM rapamycin and1.5 mCl ml-1 ³²P-orthophosphate, stimulated for 5 minutes with insulin(0.1 μM) or buffer and GSK3 isoforms co-immunoprecipitated from thelysates as in FIG. 1.

Discussion

Inhibition of GSK3 induced by insulin in L6 myotubes in FIG. 1 (a-c) wasunaffected by agents which prevented the activation of MAPKAP kinase-1(8-bromo-cyclic AMP, or PD 98059) [see Alessi et al., J Biol Chem, Vol.270, No. 46, pp. 27489-27494 (1995)], FIG. 1 (d and e) and/or p70^(S6k)rapamycin [see Kuo et al., Nature, Vol. 358, No. 6381, pp. 70-73 (1992);and Cross et al. (1994), supra], suggesting that neither MAPKAP kinase-1nor p70^(S6k) are essential for this process. However, thephosphorylation and inhibition of GSK3-β after phorbol ester treatment[see Stambolic and Woodget, Biochem J, Vol. 303, Pt. 3, pp. 701 -704(1994)] is enhanced by co-expression with MAPKAP kinase-1 in HeLa S3cells, whereas in NIH 3T3 cells the EGF-induced inhibition of GSK3-α andGSK3-β [see Saito, Vandenheede and Cohen (1994), supra] is largelysuppressed by expression of a dominant-negative mutant of MAP kineaskinase-1. See Eldar-Finkelman, Seger, Vandenheede and Krebs, J BiolChem, Vol. 270, No. 3, pp. 987-990 (1995). MAPKAP kinase-1 may thereforemediate the inhibition of GSK3 by agonists which are much more potentactivators of the classical MAP kinase pathway than is insulin.

To identify the insulin-stimulated protein kinase (ISPK) that inhibitsGSK3 in the presence of rapamycin and PD 98059, L6 myotubes wereincubated with both compounds and stimulated with insulin. The lysateswere then chromatographed on Mono Q and the fractions assayed with“Crosstide” GRPRTSSFAEG (SEQ ID NO:5), a peptide corresponding to thesequence in GSK3 surrounding the Ser (underlined) phosphorylated byMAPKAP kinase-1 and p70^(S6k) (Ser21 in GSK3-α) [see Sutherland andCohen (1994), supra] and Ser9 in GSK3-β. See Sutherland, Leighton andCohen, Biochem J, Vol. 296, Pt. 1, pp. 15-19 (1993). Three peaks ofCrosstide kinase activity were detected, which were absent if insulinstimulation was omitted or if the cells were first preincubated with thePI 3-kinase inhibitor wortmannin. See FIG. 2 (a). Wortmannin [see Crosset al. (1994),supra; and Welsh et al. (1994), supra], and thestructurally-unrelated PI 3-kinase inhibitor LY 294002; FIG. 1 (b), bothprevent the inhibition of GSK3 by insulin.

The PKs RAC-PK-α, RAC-PK-β and RAC-PKγ are Ser-/Thr-specific andcellular homologues of the viral oncogene v-akt. See Coffer andWoodgett, Eur J Biochem, Vol. 201, No. 2, pp. 475-481 (1991); Jones etal. (1991), supra, Ahmed et al., Mol Cell Biol, Vol. 15, pp. 2304-2310(1995); and Cheng et al., Proc Natl Acad Sci USA, Vol. 89, No. 19, pp.9267-9271 (1992). These enzymes have recently been shown to be activatedin NIH 3T3, Rat-1 or Swiss 3T3 cells in response to growth factors orinsulin, activation being suppressed by blocking the activation of PI3-kinase in different ways. See Franke et al., (1995), supra; andBurgering and Coffer (1995), supra. All three peaks of Crosstide kinase[see FIG. 2 (a)], but no other fraction from Mono Q, showed thecharacteristic multiple bands of RAC-PK (relative molecular mass, Mr58K, 59K or 60K) that have been observed in other cells, whenimmunoblotting was performed with an antibody raised against thecarboxyl-terminal peptide of RAC-PK-α. See FIG. 2 (b). The more slowlymigrating forms represent more highly-phosphorylated protein, and areconverted to the fastest migrating species by treatment withphosphatases. Phosphatase treatment also results in the inactivation ofRAC-PK [see Burgering and Coffer (1995), supra] and the complete loss ofCrosstide kinase activity (data not shown). Of the Crosstide kinaseactivity in peaks 2 and 3 from Mono Q, 70-80% was immunoprecipitated bya separate antibody raised against the amino-terminal PH domain ofRAC-PK-α. The C-terminal antibody also immunoprecipitated RAC-PKactivity specifically from peaks 2 and 3, but was less effective thanthe anti-PH-domain antibody. Peak-1 was hardly immunoprecipitated byeither antibody and may represent RAC-PKβ. An immunoprecipitatinganti-MAPKAP kinase-1 antibody [see Cross et al. (1994), supra] failed todeplete any of the Crosstide kinase activity associated with peaks 1, 2or 3.

Insulin stimulation of L6 myotubes increased RAC-PK activity by morethan 10-fold [see FIG. 2 (c)], and activation was blocked by wortmanninor LY 294002, but was essentially unaffected by 8-bromo-cyclic AMP orrapamycin plus PD 98059. See FIG. 2 (c). The half-time (t0.5) oractivation of RAC-PK (1 minute) was slightly faster than that forinhibition of GSK3 (2 minutes). See Cross et al. (1994), supra. Incontrast, the activation of MAPKAP kinase-1 [see FIG. 2 (d)] andp70^(s6k) (not shown) was slower (t0.5>5 minutes). Activation of MAPKAPkinase-1 was prevented by 8-bromo-cyclic AMP or PD 98059 [see FIG. 2(d)], and activation of p70^(S6k by rapamycin. See Cross et al. ()1994),supra. Akt/RAC phosphorylated the Ser in the Crosstide equivalent toSer21 in GSK3-α and Ser9 in GSK3-β (data not shown).

RAC-PK from insulin-stimulated L6 myotubes (but not from unstimulated orwortmannin-treated cells) inactivated GSK3-α and GSK3-β in vitro, andinhibition was reversed by the Ser-Thr-specific protein phosphatasePP2A. See Embi, Rylatt and Cohen (1980) and FIG. 3 (a). To furtherestablish that inactivation was catalyzed by RAC-PK, and not by aco-immunoprecipitating PK, hemagglutonin-tagged RAC-PK-α (HA-RAC-PK) wastransfected into COS-1 cells and activated by stimulation withpervanadate, which is the strongest inducer of RAC-PK activation in thissystem. The HA-RAC-PK inactivated GSK3-β, but not if treatment withpervanadate was omitted or if WT HA-RAC-PK was replaced with a “kinaseinactive” mutant. See FIG. 3 (b).

The inactivation of GSK3-β by RAC-PK in vitro was accompanied by thephosphorylation of one major tryptic peptide [see FIG. 4 (a)] whichco-eluted during C18-chromatography [See Sutherland, Leighton and Cohen,Biochem J, (1993), supra] and isoelectric focusing with that obtainedafter phosphorylation by MAPKAP kinase-1. See FIG. 4 (d). Stimulation ofL6 myotubes with insulin (in the presence of rapamycin and PD 98059)increased the ³²P-labelling of GSK3-α and GSK3-β by 60-100% [see FIG. 4(c)] and increased the ³²P-labelling of the same tryptic peptideslabelled in vitro. See FIG. 4 (d). Sequence analyses established thatthe third residue of these, corresponding to Ser9 (GSK3-β) or Ser21(GSK3-α), was the site of phosphorylation in each phosphopeptide, bothin vitro [see FIG. 4 (b)] and in vivo (not shown). The ³²P-labelling ofother (more acidic) tryptic phosphopeptides was not increased byinsulin. See FIG. 4 (d). These peptides have been noted previously inGSK3 from A431 cells and shown to contain phosphoserine andphosphotyrosine. See Saito, Vandenheede and Cohen, Vol. 303 (1994),supra.

PKC-delta (δ), epsilon (ε) and zeta (ζ) are reported to be activated bymitogens, and PKC-ζ activity is stimulated in vitro by several inositolphospholipids, including PI(3, 4, 5)P3 the product of the PI 3-kinasereaction. See Andjelkovic et al., Proc Natl Acad Sci USA, Vol. 93, No.12, pp. 5699-5704 (1995). However, purified PKC-ε [see Palmer et al., JBiol Chem, Vol. 270, No. 38, pp. 22412-22416 (1995)], PKC-ε and PKC-ζ(data not shown) all failed to inhibit GSK3-α or GSK3-β in vitro.Moreover, although PKC-α, β1 and γ inhibit GSK3-β in vitro [see Palmeret al. (1995), supra], GSK3-α is unaffected, while their downregulationin L6 myotubes by prolonged incubation with phorbol esters abolishes theactivation of MAPKAP kinase-1 in response to subsequent challenge withphorbol esters, but has no effect on the inhibition of GSK3 by insulin(not shown).

Taken together, our results identify GSK3 as a substrate for RAC-PK. Thestimulation of glycogen synthesis by insulin in skeletal muscle involvesthe dephosphorylation of Ser residues in glycogen synthase that arephosphorylated by GSK3 in vitro. See Parker, Candwell and Cohen, Eur JBiochem, Vol. 130, No. 1, pp. 227-234 (1983). Hence the 40-50%inhibition of GSK3 by insulin, coupled with a similar activation of therelevant glycogen synthase phosphatase [see Goode, Hughes, Woodgett andParker, J Biol Chem, Vol. 267, No. 24, pp. 16878-16882 (1992)], canaccount for the stimulation of glycogen synthase by insulin in skeletalmuscle [see Parker, Candwell and Cohen (1983), supra] or L6 myotubes.See Goode, Hughes, Woodgett and Parker (1992), supra. The activation ofglycogen synthase and the resulting stimulation of glycogen synthesis byinsulin in L6 myotubes is blocked by wortmannin, but not by PD 98059[see Dent et al., Nature, Vol. 348, pp. 302-308 (1990)], just like theactivation of Akt/RAC and inhibition of GSK3. However, GSK3 is unlikelyto be the only substrate of RAC-PK in vivo, and identifying otherphysiologically relevant substrates will be important because RAC-PKβ isamplified and over-expressed in many ovarian neoplasms. See Cheng et al.(1992), supra.

EXAMPLE 5

Activation of RAC-PK by Insulin in L6 Myotubes is Accompanied byPhosphorylation of Residues Thr308 and Ser473

Insulin induces the activation and phosphorylation of RAC-PKα in L6myotubes. Three 10 cm dishes of L6 myotubes were ³²P-labelled andtreated for 10 minutes with or without 100 nM wortmannin and then for 5minutes with or without 100 nM insulin. RAC-PKα was immunoprecipitatedfrom the lysates and an aliquot (15%) assayed for RAC-PKα activity. SeeFIG. 5 (a). The activities are plotted ±SEM for 3 experiments relativeto RAC-PKα derived from unstimulated cells which was 10 mU/mg. Theremaining 85% of the immunoprecipitated RAC-PKα was alkylated with4-vinylpyridine, electrophoresed on a 10% polyacrylamide gel (preparedwithout SDS to enhance the phosphorylation-induced decrease in mobility)and autoradiographed. The positions of the molecular mass markersglycogen phosphorylase (97 kDa), bovine serum albumin (66 kDa) andovalbumin (43 kDa) are marked.

Under these conditions, insulin stimulation resulted in a 12-foldactivation of RAC-PKα [see FIG. 5 (a)] and was accompanied by a1.9±0.3-fold increase in ³²P-labelling (4 experiments) and retardationof its mobility on SDS-polyacrylamide gels. See FIG. 5 (b). Theactivation of RAC-PKα, the increase in its ³²P-labelling and reductionin electrophoretic migration were all abolished by prior incubation ofthe cells with 100 nM wortmannin. Phosphoamino acid analysis of thewhole protein revealed that ³²P-labelled RAC-PKα was phosphorylated atboth serine and threonine residues and that stimulation with insulinincreased both the ³²P-labelling of both phosphoamino acids (data notshown).

FIG. 6—Insulin stimulation of L6 myotubes induces the phosphorylation oftwo peptides in RAC-PKα. Bands corresponding to ³²P-labelled RAC-PKα,from FIG. 5 (b), were excised from the gel, treated with 4-vinylpyridineto alkylate cysteine (Cys) residues, digested with trypsin andchromatographed on a Vydac 218TP54 C18-column (Separations Group,Hesperia, Calif.) equilibrated with 0.1% (by vol) trifluoroacetic acid(TFA) and the columns developed with a linear acetonitrile gradient(diagonal line). The flow rate was 0.8 mL/min. and fractions of 0.4 mLwere collected.

(a)—Tryptic peptide map of ³²P-labelled RAC-PKα from unstimulated L6myotubes.

(b)—Tryptic peptide map of ³²P-labelled RAC-PKα from insulin-stimulatedL6 myotubes.

(c)—Tryptic peptide map of ³²P-labelled RAC-PKα from L6 myotubes treatedwith wortmannin prior to insulin. The two major ³²P-labelled peptideseluting at 23.7% and 28% acetonitrile are named Peptide A and Peptide B,respectively. Similar results were obtained in 4 (a and b) and 2 (c)experiments.

No major ³²P-labelled peptides were recovered from ³²P-labelled RAC-PKαderived from unstimulated L6 myotubes [see FIG. 6 (a)] indicating that,in the absence of insulin, there was a low level phosphorylation at anumber of sites. However, following stimulation with insulin, two major³²P-labelled peptides were observed, termed A and B [see FIG. 6 (b)],whose ³²P-labelling was prevented if the myotubes were firstpreincubated with wortmannin. See FIG. 6 (c).

FIG. 7—Identification of the phosphorylation sites in Peptides A and B.

(a)—Peptides A and B from FIG. 5 (b) (1000 cpm) were incubated for 90minutes at 110° C. in 6 M HCl, electrophoresed on thin layer celluloseat pH 3.5 to resolve orthophosphate (Pi), phosphoserine (pS),phosphthreonine (pT) and phosphotyrosine (pY) and autoradiographed.

(b)—Peptide A [see FIG. 5 (b 3)] obtained from 50 10 cm dishes of³²P-labelled L6 myotubes was further purified by chromatography on amicrobore C18-column equilibrated in 10 mM ammonium acetate pH 6.5instead of 0.1% TFA. A single peak of ³²P-radioactivity was observed at21% acetonitrile which coincided with a peak of 214 nm absorbance.Eighty percent (80%) of the sample (1 pmol) was analysed on an AppliedBiosystems 476A sequencer to determine the amino acid sequence, and thephenylthiohydantoin (Pth) amino acids identified after each cycle ofEdman degradation are shown using the single letter code for aminoacids. The residues in parentheses were not present in sufficientamounts to be identified unambiguously. To identify the site(s) ofphosphorylation, the remaining 20% of the sample (600 cpm) was thencoupled covalently to a Sequelon arylamine membrane and analysed on anApplied Biosystems 470A sequencer using the modified program describedby Stokoe et al., EMBO J, Vol. 11, No.11, pp. 3985-3994 (1992).³²P-radioactivity was measured after each cycle of Edman degradation.

(c)—Peptide B from FIG. 2 (b) (800 cpm) was subjected to solid phasesequencing as in (b).

Peptide A was phosphorylated predominantly on Ser while Peptide B waslabelled on Thr. See FIG. 7(a). Amino acid sequencing established thatPeptide A commenced at residue 465. Only a single burst of³²P-radioactivity was observed after the eighth cycle of Edmandegradation [see FIG. 7 (b)], demonstrating that insulin stimulation ofL6 myotubes had triggered the phosphorylation of RAC-PKα at Ser473,which is located 9 residues from the C-terminus of the protein.Phosphopeptide B was only recovered in significant amounts if³²P-labelled RAC-PKα was treated with 4-vinylpyridine prior to digestionwith trypsin, indicating that this peptide contained a Cys residue(s),and a single burst of ³²p-radioactivity was observed after the firstcycle of Edman degradation. See FIG. 7 (c). This suggested that the siteof phosphorylation was residue 308, since it is the only Thr in RAC-PKαthat follows a Lysine (Lys) or Arginine (Arg) residue and which islocated in a tryptic peptide containing a Cys residue (at position 310).The acetonitrile concentration at which phosphopeptide B is eluted fromthe C18-column (28%) and its isoelectric point (4.0) are also consistentwith its assignment as the peptide comprising residues 308-325 ofRAC-PKα. The poor recoveries of Peptide B during further purification atpH 6.5 prevented the determination of its amino acid sequence, butfurther experiments described below using transiently transfected 293cells established that this peptide does correspond to residues 308-325of RAC-PKα.

FIG. 8—Mapping the phosphorylation sites of RAC-PKα in transientlytransfected 293 cells. Two hundred ninety-three (293) cells weretransiently transfected with DNA constructs expressing WT RAC-PKα, or ahemagglutonin (HA) epitope-tagged RAC-PKα encoding the human protein,such as HA-KD RAC-PKα, HA-473A RAC-PKα or HA-308A RAC-PKα. Aftertreatment for 10 minutes with or without 100 nM wortmannin, the cellswere stimulated for 10 minutes with or without 100 nM insulin or 50ng/mL IGF-1 in the continued presence of wortmannin. RAC-PKα wasimmunoprecipitated from the lysates and assayed, and activitiescorrected for the relative levels of expression of each HA-RAC-PKα. Theresults are expressed relative to the specific activity of WT HA-RAC-PKαfrom unstimulated 293 cells (2.5±0.5 U/mg).

(b)—Twenty (20) μg of protein from each lysate was electrophoresed on a10% SDS/polyacrylamide gel and immunoblotted using monoclonalHA-antibody. The molecular markers are those used in FIG. 5 (b).

FIG. 9—IGF-1 stimulation of 293 cells induces the phosphorylation of twopeptides in transfected HA-RAC-PKα. Two hundred ninety-three (293) cellstransiently transfected with WT HARAC-PKα DNA constructs were³²P-labelled, treated for 10 minutes without (a and b) or with (c) 100nM wortmannin and then for 10 minutes without (a) or with (b and c) 50ng/mL IGF-1. The ³²p-labelled HA-RAC-PKα was immunoprecipitated from thelysates, treated with 4-vinylpyridine, electrophoresed on a 10%polyacrylamide gel, excised from the gel and digested with trypsin.Subsequent chromatography on a C18-column resolved 4 majorphosphopeptides termed C, D, E and F. Similar results were obtained in 6separate experiments for (a) and (b), and in 2 experiments for (c).

Stimulation with insulin and IGF-1 resulted in 20-fold and 46-foldactivation of transfected RAC-PKα, respectively [see FIG. 8 (a)], thehalf time for activation being 1 minute, as found with other cells.Activation of RAC-PKα by insulin or IGF-1 was prevented by priorincubation with wortmannin [see FIG. 8 (a)] and no activation occurredif 293 cells were transfected with vector alone and then stimulated withinsulin or 1 GF-1 (data not shown).

Two prominent ³²P-labelled peptides were present in unstimulated 293cells. See FIG. 9 (a). One, termed Peptide C, usually eluted as adoublet at 20-21% acetonitrile while the other, termed Peptide F, elutedat 29.7% acetonitrile. Stimulation with insulin or IGF-1 did not affectthe ³²P-labelling of Peptides C and F [see FIG. 9 (a and b)], butinduced the ³²P-labelling of 2 new peptides, termed D (23.4%acetonitrile) and E (28% acetonitrile), which eluted at the sameacetonitrile concentrations as Peptides A and B from L6 myotubes [seeFIG. 6 (b)] and had the same isoelectric points (7.2 and 4.0,respectively). Treatment of 293 cells expressing HA-RAC-PKα with 100 nMwortmannin, prior to stimulation with IGF-1, prevented thephosphorylation of Peptides D and E, but had no effect on the³²P-labelling of Peptides C and F. See FIG. (c).

Peptides C, D, E and F were further purified by re-chromatography on theC18-column at pH 6.5 and sequenced. Peptides C gave rise to threeseparate (but closely eluting) ³²P-labelled peptides (data not shown).Amino acid sequencing revealed that all 3 commenced at residue 122 ofRAC-PKα and that Ser124 was the site of phosphorylation. See FIG. 10(a). Peptide D only contained phosphoserine and, as expected,corresponded to the RAC-PKα tryptic peptide commencing at residue 465that was phosphorylated at Ser473. See FIG. 10 (b). Peptide E, onlycontained phosphothreonine and amino acid sequencing demonstrated thatit corresponded to residues 308-325, the phosphorylation site beingThr308. See FIG. 10 (c). Peptide F only contained phosphothreonine andcorresponded to the peptide commencing at residue 437 of RAC-PKαphosphorylated at Thr450. See FIG. 10 (d).

In the presence of phosphatidylserine, RAC-PKα binds to PIP3 withsubmicromolar affinity. See James et al., Biochem J, Vol. 315, Pt. 3,pp. 709-713 (1996); and Frech, Andjelkovic, Falck and Hemmings, inpreparation (1996). Phosphatidyl 4,5-bisphosphate and phosphatidyl 3,4bisphosphate bind to RAC-PKα with lower affinities and PI 3,5bisphosphate and PI 3 phosphate did not bind at all under theseconditions. See James et al. (1996), supra. The region of RAC-PKα thatinteracts with PIP3 is almost certainly the PH domain, because theisolated PH domain binds PIP3 with similar affinity to RAC-PKα itself[see Frech, Andjelkovic, Falck and Hemmings (1996), supra] and becausethe PH domain of several other proteins, such as the PH-domains of,β-spectrin and phospholipase Cl, are known to interact specifically withother phosphoinositides. See Hyvonen et al., EMBO J, Vol. 14, No. 19,pp. 4676-4685 (1995); and Lemmon et al., Proc Natl Acad Sci USA, Vol.92, No. 23, pp. 10472-10476 (1995).

The experiments described above were repeated using insulin instead ofIGF-1. The results were identical, except that the ³²P-labelling ofPeptides D and E was about 50% of the levels observed with IGF-1 (datanot shown). This is consistent with the two-fold lower level ofactivation of RAC-PKα by insulin compared with IGF-1 (FIG. 7A).

EXAMPLE 6

MAPKAP Kinase-2 Phosphorylates Ser473 of RAC-PKα Causing PartialActivation

Ser473 of RAC-PKα lies in a consensus sequencePhe-x-x-Phe/Tyr-SerfThr-Phe/Tyr found to be conserved in a number of PKsthat participate in signal transduction pathways. See Pearson et al.,EMBO J, Vol. 14, No. 21, pp. 5279-5287 (1995). In order to Identify theSer473 kinase(s) we therefore chromatographed rabbit skeletal muscleextracts on CM-Sephadex, and assayed the fractions for protein kinasescapable of phosphorylating a synthetic peptide corresponding to residues465-478 of RAC-PKα. These studies identified an enzyme eluting at 0.3 MNaCl which phosphorylated the peptides 465-478 at the residue equivalentto Ser473 of RAC-PKα. The Ser473 kinase co-eluted from CM-Sephadex withMAPKAP kinase-2 [see Stokoe et al. (1992), supra], which is a componentof a stress and cytokine-activated MAP kinase cascade. See Rouse et al.,Cell, Vol. 78, No. 6, pp. 1027-1037 (1994); and Cuenda et al., FEBSLett, Vol. 364, No. 2, pp. 229-233 (1995). The Ser473 kinase continuedto cofractionate with MAPKAP kinase-2 through phenyl-Sepharose,heparin-Sepharose, Mono S and Mono Q and was immunoprecipitatedquantitatively by an anti-MAPKAP kinase-2 antibody [see Gould, Cuenda,Thomson and Cohen, Biochem J, Vol. 311, pp. 735-738 (1995)]demonstrating that MAPKAP kinase-2 was indeed the Ser473 kinase we hadpurified.

FIG. 11—HA-RAC-PKα was immunoprecipitated from the lysates ofunstimulated COS-1 cells expressing these constructs.

(a)—0.5 μg of immunoprecipitated HA-RAC-PKα was incubated with MAPKAPkinase-2 (50 U/mL), 10 mM magnesium acetate and 100 mM [γ³²P]ATP in atotal of 40 μL of Buffer B. At various times, aliquots were removed andeither assayed for RAC-PKα activity (open circles) or for incorporationof phosphate into RAC-PKα (closed circles). Before measuring RAC-PKaactivity, EDTA was added to a final concentration of 20 mM to stop thereaction, and the immunoprecipitates washed twice with 1.0 mL of bufferB containing 0.5 M NaCl, then twice with 1.0 mL of buffer B to removeMAPKAP kinase-2. The results are presented as ±SEM for 6 determinations(2 separate experiments) and RAC-PKα activities are presented relativeto control experiments in which HA-RAC-PKα was incubated with MgATP inthe absence of MAPKAP kinase-2 (which caused no activation).Phosphorylation was assessed by counting the ³²P-radioactivityassociated with the band of RAC-PKα after SDS/polyacrylamide gelelectrophoresis. The open triangles show the activity ofimmunoprecipitated HA-KD RAC-PKα phosphorylated by MAPKAP kinase-2.

(b)—HA-RAC-PKα phosphorylated for 1 hour with MAPKAP kinase-2 and32P-γ-ATP as in (a) was digested with trypsin and chromatographed on aC18-column as described in the legend for FIG. 2 (c). The major³²P-labelled peptide from (b) was analyzed on the 470A sequencer as inFIG. 3 to identify the site of phosphorylation.

Bacterially-expressed MAPKAP kinase-2 phosphorylated WT HA-RAC-PKα orthe catalytically-inactive mutant HA-RAC-PKα in which Lys179 had beenmutated to Ala (data not shown) to a level approaching 1 mol per moleprotein. See FIG. 11 (a). Phosphorylation of WT RAC-PKα was paralleledby a 7-fold increase in activity, whereas phosphorylation of thecatalytically-inactive mutant did not cause any activation. See FIG. 11(a). No phosphorylation or activation of WT HA-RAC-PKα occurred ifMAPKAP kinase-2 or MgATP was omitted from the reaction (data not shown).WT HA-RAC-PKα that had been maximally-activated with MAPKAP kinase-2,was completely dephosphorylated and inactivated by treatment withprotein phosphatase 2A (data not shown).

HA-RAC-PKα that had been maximally-phosphorylated with MAPKAP kinase-2was digested with trypsin and C18-chromatography revealed a single major³²P-labelled phosphoserine-containing peptide. See FIG. 11 (b). Thispeptide eluted at the same acetonitrile concentration [see FIG. 11 (b)]and had the same isoelectric point of 7.2 (data not shown) as the³²P-labelled tryptic peptide containing Ser473 [compare FIG. 11 (b) andFIG. 6 (b)]. Solid phase sequencing gave a burst of ³²P-radioactivityafter the eighth cycle of Edman degradation [see FIG. 11 (c)],establishing that Ser473 was the site of phosphorylation. The same³²P-peptide was obtained following tryptic digestion of catalyticallyinactive HA-KD RAC-PKα that had been phosphorylated with MAPKAP kinase-2(data not shown).

EXAMPLE 7

Phosphorylation of Thr308 and Ser473 Causes Synergistic Activation ofRAC-PKα

The experiments described above demonstrated that phosphorylation ofSer-473 activates RAC-PKα in vitro but did not address the role ofphosphorylation of Thr-308, or how phosphorylation of Thr-308 mightinfluence the effect of Ser-473 phosphorylation on activity, or viceversa. We therefore prepared HA-tagged RAC-PKα DNA constructs in whicheither Ser473 or Thr308 would be changed either to Ala (to block theeffect of phosphorylation) or to Asp (to try and mimic the effect ofphosphorylation).

FIG. 12—Activation of HA-RAC-PKα mutants in vitro by MAPKAP kinase-2.

(a)—WT and mutant HA-RAC-PKα proteins were immunoprecipitated from thelysates of unstimulated COS-1 cells expressing these constructs andincubated for 60 minutes with MgATP in the absence (−, filled bars) orpresence (+, hatched bars) of MAPKAP kinase-2 and MgATP (50 U/mL). TheRAC-PKα protein was expressed as similar levels in each construct andspecific activities are presented relative to WT HA-RAC-PKα incubated inthe absence of MAPKAP kinase-2 (0.03 U/mg). The results are shown as theaverage ±SEM for 3 experiments.

(b)—Twenty (20) μg of protein from each lysate was electrophoresed on a10% SDS/polyacrylamide gel and immunoblotted using monoclonalHA-antibody.

All the mutants were expressed at a similar level in serum-starved COS-1cells (data not shown) and the effects of maximally phosphorylating eachof them at Ser473 is shown in FIG. 12 (a). Before phosphorylation withMAPKAP kinase-2 the activity of HA-473A RAC-PKα was similar to that ofunstimulated WT HA-RAC-PKα and, as expected, incubation with MAPKAPkinase-2 and MgATP did not result in any further activation of HA-473ARAC-PKα. In contrast, the activity of HA-473D RAC-PKα was 5- to 6-foldhigher than that of unstimulated WT HARAC-PKα protein, and similar tothat of WT HA-RAC-PKα phosphorylated at Ser473. As expected, HA-473DRAC-PKα was also not activated further by incubation with MAPKAPkinase-2 and MgATP. The activity of HA-308A RAC-PKα was about 40% thatof the unstimulated WT enzyme, and after phosphorylation with MAPKAPkinase-2 is activity increased to a level similar to that of WTHA-RAC-PKα phosphorylated at Ser473. Interestingly, HA-308D RAC-PKαwhich (like HA473D PK) was 5-fold more active than dephosphorylated WTHA-RAC-PKα, was activated dramatically by phosphorylation of Ser473.After incubation with MAPKAP kinase-2 and MgATP, the activity of HA-308DRAC-PKα was nearly 5-fold higher than that of WT HA-RAC-PKαphosphorylated at Ser473. See FIG. 12 (b). These results suggested thatthe phosphorylation of either Thr308 or Ser473 leads to partialactivation of RAC-PKα in vitro, and that phosphorylation of bothresidues results in a synergistic activation of the enzyme. This ideawas supported by further experiments in which both Thr308 and Ser473were changed to Asp. When this double-mutant was expressed in COS-1cells it was found to possess an 18-fold higher specific activity thanthe dephosphorylated WT protein. As expected, the activity of thismutant was not increased further by incubation with MAPKAP kinase-2 andMgATP. See FIG. 12 (b).

EXAMPLE 8

Phosphorylation of Both Thr308 and Ser473 is Required for a High Levelof Activation of RAC-PKα In Vivo

FIG. 9—Effect of mutation of RAC-PKα on its activation by insulin in 293cells.

(a)—Two hundred ninety-three (293) cells were transiently transfectedwith DNA constructs expressing WT RAC-PKα, HA-D473-RAC-PKα andHA-308D/473D-RAC-PKα. After treatment for 10 minutes with or without 100nM wortmannin, cells were stimulated for 10 minutes with or without 100nM insulin in the continued presence of wortmannin. RAC-PKα wasimmunoprecipitated from the lysates and assayed, and activitiescorrected for the relative levels of HA-RAC-PKα expression as describedin the methods. The results are expressed relative to the specificactivity of WT HA-RAC-PKα obtained from unstimulated 293 cells.

(b)—Twenty (20) μg of protein from each lysate was electrophoresed on a10% SDS/polyacrylamide gel and immunoblotted using monoclonalHA-antibody.

The basal level of activity of HA-473A RAC-PKα derived from unstimulatedcells was similar to that of WT RAC-PKα. See FIG. 8 (a). Stimulation of293 cells expressing HA-473A RAC-PKα with insulin or IGF-1 increased theactivity of this mutant 3- and 5-fold, respectively; i.e., to 15% of theactivity of WT HA-RAC-PKα which had been transiently-expressed andstimulated under identical conditions. The basal activity of HA-308ARAC-PKα in unstimulated cells was also similar to that of WT HA-RAC-PKαderived from unstimulated cells, but virtually no activation of thismutant occurred following stimulation of the cells with insulin orIGF-1. These data are consistent with in vitro experiments and indicatethat maximal activation of RAC-PKα requires phosphorylation of bothSer473 and Thr308 and that phosphorylation of both residues results in asynergistic activation of the enzyme. Consistent with these results,HA-473D RAC-PKα displayed 5-fold higher activity and the HA-308D/HA473Ddouble-mutant 40-fold higher activity than WT HA-RAC-PKα when expressedin unstimulated cells. Following stimulation with insulin, HA-473DRAC-PKA was activated to a level similar to that observed with the WTenzyme, while the HA-308D/HA-473D double-mutant could not be activatedfurther. See FIG. 13. As expected, activation of HA-473D RAC-PKα byinsulin was prevented by wortmannin, and the activity of the HA-308D/HA-473D double-mutant was resistant to wortmannin. See FIG. 13.

EXAMPLE 9

Phosphorylation of Thr308 is Not Dependent on Phosphorylation of Ser473or Vice Versa (in 293 Cells)

FIG. 10—A 10 cm dish of 293 cells were transfected with either HA-308ARAC-PKα or HA-473A RAC-PKα, ³²P-labelled, then stimulated for 10 minuteswith either IGF-1 (50 ng/mL) or buffer. The ³²P-labelled RAC-PKα mutantswere immunoprecipitated from the lysates, treated with 4-vinylpyridine,electrophoresed on a 10% polyacrylamide gel, excised from the gel anddigested with trypsin, then chromatographed on a C18-column. The trypticpeptides containing the phosphorylated residues Ser124, Thr308, Thr450,Ser473 are marked and their assignments were confirmed by phosphoaminoacid analysis and sequencing to identify the sites of phosphorylation(data not shown). The phosphopeptides containing Thr308 and Ser473 wereabsent if stimulation with IGF-1 was omitted, while the phosphopeptidescontaining Ser124 and Thr450 were present at similar levels as observedwith WT RAC-PKα. See FIG. 9 (a). Similar results were obtained in 3separate experiments.

These experiments demonstrated that IGF-1 stimulation induced thephosphorylation of HA-473A RAC-PKα at Thr308, and the phosphorylation ofHA-308A RAC-PKα at Ser473. Similar results were obtained afterstimulation with insulin rather than IGF-I.

EXAMPLE 10

IGF-1 or Insulin Induces Phosphorylation of Thr308 and Ser473 in aCatalytically Inactive Mutant of RAC-PKα

FIG. 15—The catalytically-inactive RAC-PKα mutant (HA-KD-RAC-PKα)expressed in 293 cells is phosphorylated at Thr308 and S er473 afterstimulation with IGF-1. Each 10 cm dish of 293 cellstransiently-transfected with HA-KD-RAC-PKα DNA constructs was³²P-labelled and incubated for 10 minutes with buffer (a), 50 ng/mLIGF-1 (b) or 100 nM insulin (c). The ³²P-labelled HA-KD-RAC-PKα wasimmunoprecipitated from the lysates, treated with 4 vinylpyridine,electrophoresed on a 10% polyacrylamide gel, excised from the gel anddigested with trypsin, then chromatographed on a C18-column. The trypticpeptides containing the phosphorylated residues Ser124, Thr308, Thr450and Ser473 are marked. Similar results were obtained in 3 separateexperiments for (b) and (b), and in 2 experiments for (c).

This “kinase dead” mutant of RAC-PKα, termed HA-KD-RAC-PKα, in whichLys179 was changed to Ala (see above) was transiently expressed in 293cells and its level of expression found to be several-fold lower thanthat of WT HA-RAC-PKα expressed under identical conditions. See FIG. 8(b). As expected, no RAC-PKα activity was detected when 293 cellsexpressing HA-KD-RAC-PKα, were stimulated with insulin or IGF-1. SeeFIG. 7 (a).

Two hundred ninety-three (293) cells that had been transientlytransfected with HA-KD-RAC-PKα were ³²P-labelled, then stimulated withbuffer, insulin or IGF-1 and sites on RAC-PKα phosphorylated under theseconditions were mapped. In contrast to WT HA-RAC-PKα from unstimulated293 cells (see FIG. 9), HA-KD RAC-PKα was phosphorylated to a much lowerlevel at Ser124, but phosphorylated similarly at Thr450. See FIG. 15(a). Following stimulation with IGF-1 [see FIG. 15 (b)] or insulin [seeFIG. 14 (c)], HA-KD-RAC-PKα became phosphorylated at the peptidescontaining Thr308 and Ser473, the extent of phosphorylation of thesesites being at least as high as WT RAC-PKα. Amino acid sequencing ofthese peptides established that they were phosphorylated at Thr308 andSer473, respectively.

The above examples establish that RAC-PK influences GSK3 activity; thatThr308 and Ser473 are the major residues in RAC-PKα that becomephosphorylated in response to insulin or IGF-1 (see FIGS. 2 and 5) andthat phosphorylation of both residues is required to generate a highlevel of RAC-PKα activity. Thus, mutation of either Thr308 or Ser473 toAla greatly decreased the activation of transfected RAC-PKα by insulinor IGF-1 in 293 cells. See FIG. 8. Moreover, RAC-PKα became partiallyactive in vitro when either Thr308 or Ser473 were changed to Asp or whenSer473 was phosphorylated by MAPKAP kinase-2 in vitro, and far moreactive when the D308 mutant of RAC-PKα was phosphorylated by MAPKAPkinase-2 or when Thr308 and Ser473 were both mutated to Asp. See FIG.12. Moreover, the D308/D473 double-mutant could not be activated furtherby stimulating cells with insulin. See FIG. 13. These observationsdemonstrate that the phosphorylation of Thr308 and Ser473 actsynergistically to generate a high level of RAC-PKα activity.

Thr308, and the amino acid sequence surrounding it, is conserved in ratRAC-PKβ and RAC-PKγ but, interestingly, Ser473 (and the sequence surrounding it) is only conserved in RAC-PKβ. In rat RAC-PKγ, Ser473 ismissing because the C-terminal 23 residues are deleted. This suggeststhat the regulation of RAC-PKγ may differ significantly from that ofRAC-PKα and RAC-PKβ in the rat.

Thr308 is located in subdomain VIII of the kinase catalytic domain, 9residues upstream of the conserved Ala-Pro-Glu motif, the same positionas activating phosphorylation sites found in many other PKs. However,Ser473 is located C-terminal to the catalytic domain in the consensussequence Phe-Xaa-Xaa-Phe/Tyr-Ser/Thr-Phe/Tyr which is present in severalprotein kinases that participate in growth factor-stimulated kinasecascades, such as p70 S6 kinase, PKC and p9orsk. See Pearson et al.(1995), supra. However, it is unlikely that a common PK phosphorylatesthis motif in every enzyme for the following reasons. Firstly,phosphorylation of the equivalent site in p70 S6 kinase is prevented bythe immunosuppressant drug rapamycin [see Pearson et al. (1995), supra]which does not prevent the activation of RAC-PKα by insulin [see Crosset al., Nature, Vol. 378, No. 6559, pp. 785-789 (1995)] or isphosphorylation at Ser473. See D. Alessi, unpublished work. Secondly,the equivalent residue in PK cascade is phosphorylated constitutivelyand not triggered by stimulation with growth factors. See Tsutakawa etal., J Biol Chem, Vol. 270, No. 45, pp. 26807-26812 (1995).

MAPKAP kinase-2 is a component of a PK cascade which becomes activatedwhen cells are stimulated with interleukin-1 or tumour necrosis factoror exposed cellular stresses. See Rouse et al. (1994), supra; and Cuendaet al. (1995), supra. MAPKAP kinase-2 phosphorylates RAC-PKαstoichiometrically at Ser473 (see FIG. 11) and this finding was usefulin establishing the role of Ser473 phosphorylation in regulating RAC-PKαactivity. However, although MAPKAP kinase-2 activity is stimulated to asmall extent by insulin in L6 cells, no activation could be detected in293 cells in response to insulin or IGF-1. Moreover, exposure of L6cells or 293 cells to a chemical stress (0.5 mM sodium arsenite)strongly activated MAPKAP kinase-2 (see D. Alessi, unpublished work) asfound in other cells [see Rouse et al. (1994), supra; and Cuenda et al.(1995), supra], but did not activate RAC-PKα at all. Furthermore, thedrug SB 203580 which is a specific inhibitor of the PK positionedimmediately upstream of MAPKAP kinase-2 [see Cuenda et al. (1995),supra], prevented the activation of MAPKAP kinase-2 by arsenite but hadno effect on the activation of RAC-PKα by insulin or IGF-1. Finally, theactivation of RAC-PKα was prevented by wortmannin (see FIGS. 6 and 9),but wortmannin had no effect on the activation of MAPKAP kinase-2 in L6or 293 cells. It should also be noted that the sequence surroundingSer473 of RAC-PKα (HFPQFSY) does not conform to the optimal consensusfor phosphorylation by MAPKAP kinase-2 which requires Arg at positionn-3 and a bulky hydrophobic residue at position n-5, where n is theposition of the phosphorylated residue. The Km for phosphorylation ofthe synthetic peptide comprising residues 465-478 is nearly 100-foldhigher than the Km for the standard MAPKAP kinase-2 substrate peptide(data not shown). It is therefore unlikely that MAPKAP kinase-2 mediatesthe phosphorylation of Ser473 in vivo.

The enzyme(s) which phosphorylates Thr308 and Ser473 in vivo does notappear to be RAC-PKα itself. Thus incubation of the partially activeAsP-308 mutant with MgATP did not result in the phosphorylation ofSer473, phosphorylation of the latter residue only occurring when MAPKAPkinase-2 was added. See FIG. 11 (a) and FIG. 12. Similarly, Thr308 didnot become phosphorylated when either the partially-active D473 mutantor the partially-active Ser473 phosphorylated form of RAC-PKα wereincubated with M gATP. RAC-PKα when bound to lipid vesicles containingphosphatidylserine and PIP3 also fails to activate upon incubation withMgATP [see Alessi et al. (1996), supra] and after transfection into 293cells, a “kinase dead” mutant of RAC-PKα became phosphorylated on Thr308and Ser473 in response to insulin or IGF-1. See FIG. 14. Furthermore,HA-RAC-PKα from either unstimulated or insulin-stimulated 293 cellsfailed to phosphorylate the synthetic C-terminal peptide comprisingamino acids 467-480.

In unstimulated L6 myotubes, the endogenous RAC-PKα was phosphorylatedat a low level at a number of sites [see FIG. 6 (a)], but inunstimulated 293 cells the transfected enzyme was heavily phosphorylatedat Ser124 and Thr450. See FIG. 10. Ser124 and Thr450 are both followedby praline (Pro) residues suggesting the involvement of “Pro-directed”PKs. Although, the phosphorylation of Ser124 was greatly decreased when“kinase dead” RAC-PKα was transfected into 293 cells (see FIG. 14), itwould be surprising if Ser124 is phosphorylated by RAC-PKα itselfbecause the presence of a C-terminal Pro abolishes the phosphorylationof synthetic peptides by RAC-PKα (see D. Alessi, unpublished work).Since transfected RAC-PKα is inactive in unstimulated 293 cells (seeFIG. 12), phosphorylation of Ser124 and Thr450 clearly does not activateRAC-PKα directly. Ser 24 is located in the linker region between the PHdomain and the catalytic domain of the mammalian RAC-PKα isoforms but,unlike Thr450, is not conserved in the Drosophila homologue. SeeAndjelkovic et al., Proc Nat Acad Sci USA (1995), supra.

While we do not wish to be bound by hypotheses, the results describedsuggest that agonists which activate PI 3-kinase are likely to stimulateRAC-PKα activity via one of the following mechanisms. Firstly, PIP3 orP13,4-bisP may activate one or more protein kinases which thenphosphorylate RAC-PKα at Thr308 and Ser473. Secondly, the formation ofPIP3 may lead to the recruitment of RAC-PKα to the plasma membrane whereit is activated by a membrane-associated PK(s). The membrane associatedThr308 and Ser473 kinases might themselves be activated by PIP3 and thepossibility that Thr308 and/or Ser473 are phosphorylated directly by PI3-kinase has also not been excluded, because this enzyme is known tophosphorylate itself [see Dhand et al., EMBO J, Vol.13, No. 3, pp.522-533 (1994)] and other proteins [see Lam et al., J Biol Chem, Vol.269, No. , pp. 20648-20652 (1994)] on serine residues.

EXAMPLE 11

Molecular Basis for Substrate Specificity of RAC-PK

RAC-PKα has been shown to influence GSK3 activity. GSK3α and GSK3β arephosphorylated at Ser21 and Ser9, respectively, by 2 otherinsulin-stimulated PKs, namely p70 S6 kinase and MAP kinase-activatedPK-1 (MAPKAP-K1, also known as p90 S6 kinase). However, these enzymesare not rate-limiting for the inhibition of GSK3 by insulin in L6myotubes because specific inhibitors of their activation (rapamycin-p70S6 kinase; PD 98059-MAPKAP kinase-1) have no effect. See Cross et al.(1995), supra.

The activation of PI 3-kinase is essential for many of the effects ofinsulin and growth factors, including the stimulation of glucosetransport, fatty acid synthesis and DNA synthesis, protection of cellsagainst apoptosis and actin cytoskeletal rearrangements. Reviewed inCarpenter and Cantley, Curr Opinion Cell Biol, Vol. 8, No. 2, pp.153-158 (1996). These observations raise the question of whether RAC-PKαmediates any of these events by phosphorylating other proteins. Toaddress this issue we characterized the substrate specificityrequirements of RAC-PKα. We find that the optimal consensus sequence forphosphorylation by RAC-PKα is the motif Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hy,where Yaa and Zaa are small amino acids (other than Gly) and Hyd is alarge hydrophobic residue, such as Phe or Leu. We also demonstrate thatRAC-PKα phosphorylates histone H2B (a substrate frequently used to assayRAC-PKα in vitro) at Ser36 which lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hydmotif. These studies identified a further RAC-PKα substrate(Arg-Pro-Arg-Ala-Ala-Thr-Phe) that, unlike other peptides, is notphosphorylated to a significant extent by either p70 S6 kinase orMAPKAP-K1.

Results

Preparation of PK-Bα

In order to examine the substrate specificity of RAC-PKα, it was firstnecessary to obtain a kinase preparation that was not contaminated withany other PK activities. Two hundred ninety-three (293) cells weretherefore transiently-transfected with a DNA construct expressingHA-tagged RAC-PKα, stimulated with IGF-1 and the HA-RAC-PKαimmunoprecipitated from the lysates. IGF-1 stimulation resulted in a38-fold activation of RAC-PKα (see FIG. 16) and analysis of theimmunoprecipitates by SDS-polyacrylamide gel electrophoresis revealedthat the 60 kDa RAC-PKα was the major protein staining with coomassieBlue apart from the heavy- and light-chains of the HA monoclonalantibody. See FIG. 16, Lanes 2 and 3. The minor contaminants werepresent in control immunoprecipitates derived from 293 cells transfectedwith an empty pCMV5 vector but lacked HA-RAC-PK activity. See FIG. 16,Lane 4. Furthermore, a catalytically inactive mutant HA-RAC-PKαimmunoprecipitated from the lysates of IGF-1 stimulated 293 cells had noCrosstide kinase activity. See Alessi et al. (1996), supra. Thus, allthe Crosstide activity in HA-RAC-PK immunoprecipitates is catalyzed byRAC-PKα.

Identification of the residues in histone H2B phosphorylated by RAC-PKα.Currently, 3 substrates are used to assay RAC-PKα activity in differentlaboratories, histone H2B, MBP and Crosstide. RAC-PKα phosphorylatedCrosstide with a Km of 4 μM and a Vmax of 260 U/mg (see Table 7.1 A,peptide 1), histone H2B with a Km of 5 μM and a Vmax of 68 U/mg and MBPwith a Km of 5 μM and a Vmax of 25 U/mg. Thus the Vmax of histone H2Band MBP are 4- and 10-fold lower than for Crosstide. In order toidentify the residue(s) in histone H2B phosphorylated by RAC-PKα,³²P-labelled histone H2B was digested with trypsin (see Methods) and theresulting peptides chromatographed on a C18-column at pH 1.9. Only onemajor ³²P-labelled peptide (termed T1) eluting at 19.5% acetonitrile wasobserved. See FIG. 17 (a). The peptide contained phosphoserine (data notshown), its sequence commenced at residue 34 of histone H2B and a singleburst of radioactivity occurred after the third cycle of Edmandegradation [see FIG. 17 (b)], demonstrating that RAC-PKα phosphorylateshistone H2B at Ser36 within the sequence Arg-Ser-Arg-Lys-Glu-Ser-Tyr.Thus, like the serine phosphorylated in Crosstide, Ser36 of histone H2Blies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif (where Hyd is a bulkyhydrophobic residue-Phe in Crosstide, Tyr in H2B).

Molecular Basis for the Substrate Specificity of RAC-PKα

To further characterize the substrate specificity requirements forRAC-PKα, we first determined the minimum sequence phosphorylatedefficiently by RAC-PKα by removing residues sequently from theC-terminal and N-terminal end of Crosstide. Removal of the N-terminalGly and up to 3 residues from the C-terminus had little effect on thekinetics of phosphorylation by RAC-PKα. See Table 7.1A, comparingpeptides 1 and 5. However any further truncation of either the N- orC-terminus virtually abolished phosphorylation. See Table 7.1A, peptides8 and 9. The minimum peptide phosphorylated efficiently by RAC-PKα(Arg-Pro-Arg-Thr-Ser-Ser-Phe) was found to be phosphorylated exclusivelyat the second Ser residue as expected. Consistent with this finding, apeptide in which this Ser was changed to Ala was not phosphorylated byRAC-PKα. See Table 7.1A, peptide 7. All further studies were thereforecarried out using variants of peptide 5 in Table 7.1A (see below).

A peptide in which the second Ser of peptide 5 (see Table 7.1 A) wasreplaced by Thr was phosphorylated with a Km of 30 μM and an unchangedVmax. See Table 7.1, peptide 6. All the ³²P-radioactivity incorporatedwas present as phosphothreonine and solid phase sequencing revealed thatthe peptide was only phosphorylated at the second Thr residue, asexpected (data not shown). Thus RAC-PKα is capable of phosphorylatingThr, as well as Ser residues, but has a preference for Ser.

We next changed either of the two Arg residues in peptide 5 to Lys.These substitutions drastically decreased the rate of phosphorylation byRAC-PKα (see Table 7.1A, peptides 10 and 11), demonstrating arequirement for Arg (and not simply any basic residue) at bothpositions.

We then examined the effect of substituting the residues situatedimmediately C-terminal to the phosphorylated Ser in peptide 5, Table 7.1B. The data clearly demonstrate that the presence of a large hydrophobicresidue at this position is critical for efficient phosphorylation, withthe Km increasing progressively with decreasing hydrophobicity of theresidue at this position. See Table 7.1 B, peptides 14. Replacement ofthe C-terminal residue with Lys increased the Km 18-fold and asubstitution at this position with either Glu or praline (Pro) almostabolished phosphorylation. See Table 7.1B, peptides 5-7.

Replacement of the Thr situated 2 residues N-terminal to thephosphorylated Ser increased the Km with any amino acid tested. SeeTable 7.1C. Substitution with Ala only increased Km by 2- to 3-fold, butsubstitution with other residues was more deleterious and with Asn (aresidue of similar size and hydrophilicity to Thr) phosphorylation wasalmost abolished. See Table 7.1C. Replacement of the Ser situated 1residue N-terminal to the phosphorylated Ser also increased the Km withany amino acid tested, but the effects were less severe than at positionn-2. See Table 7.1C. When residues n-2 and n-1 were both changed to Ala,the resulting peptide RPRAASF (SEQ ID NO: 8) was phosphorylated byRAC-PKα with a Km only 5-fold higher than RPRTSSF (SEQ ID NO: 9). Incontrast the peptides RPRGGSF (SEQ ID NO: 10), RPRAGSF (SEQ ID NO: 11)and RPRGASF (SEQ ID NO: 12) were phosphorylated less efficiently. SeeTable 7.1C.

Comparison of the substrate specificity of RAC-PKα with MAPKAP kinase-1,and p70 S6 kinase. Since MAPKAP-K1 and p70 S6 kinase phosphorylate thesame residue in GSK3 phosphorylated by RAC-PKα, and studies withsynthetic peptides have established that MAPKAP-K1 and p70 S6 kinasealso preferentially phosphorylate peptides in which basic residues arepresent at positions n-3 and n-5 [see Leighton et al., FEBS Lett, Vol.375, No. 3, pp. 289-293 (1995)], we compared the specificities ofMAPKAP-K1, p70 S6 kinase and RAC-PKα in greater detail.

MAPKAP kinase-1 and p70 S6 kinase phosphorylate the peptides KKKNRTLSVA(SEQ ID NO: 13) and KKRNRTLSVA (SEQ ID NO: 14) with extremely low Kmvalues of 0.2-3.3 μM, respectively. See Table 7.2. However, thesepeptides were phosphorylated by RAC-PKα with 50- to 900-fold higher Kmvalues. See Table 7.2A, peptides 1 and 2. The peptide KKRNRTLTV (SEQ IDNO: 15), which is a relatively specific substrate for p70 S6 kinase [seeLeighton et al. (1995), supra] was also phosphorylated very poorly byRAC-PKα. See Table 7.2A, peptide 4.

Crosstide is phosphorylated by p70 S6 kinase and MAPKAP kinase-1 withsimilar efficiency to RAC-PKα. See Leighton et al. (1995), supra; Table7.2B, peptide 1; and FIG. 18. However, truncation of Crosstide togenerate the peptide RPRTSSF (SEQ ID NO: 9) was deleterious forphosphorylation by MAPKAP-K1 and even worse for p70 S6 kinase. See Table7.2B, peptides 1 and 2; and FIG. 18. Moreover, changing thephosphorylated Ser in RPRTSSF (SEQ ID NO: 9) to Thr increased the Km forphosphorylation by p70 S6 kinase much more than for RAC-PKα and almostabolished phosphorylation by MAPKAP-K1. See Table 7.2B, peptide 3; andFIG. 18. The peptide RPRAASF (SEQ ID NO: 8), was phosphorylated byMAPKAP-K1 with essentially identical kinetics to that of RAC-PKα;however phosphorylation by p70 S6 kinase was virtually abolished. SeeTable 7.2B, peptide 4; and FIG. 18. Based on these observations wesynthesized the peptide RPRAATF (SEQ ID NO: 16). This peptide wasphosphorylated by RAC-PKα with a Km of 25 μM and similar Vmax to RPRTSSF(SEQ ID NO: 9), but was not phosphorylated to a significant extent byeither MAPKAP-K1 or p70 S6 kinase. See Table 7.2B, peptide 5; and FIG.18. In FIG. 18, the PK concentration in the assays towards Crosstidewere 0.2 U/mL, and each peptide substrate was assayed at a concentrationof 30 μM. Filled bars denote RAC-PKα activity, hatched bars MAPKAPkinase-1 activity, and grey bars p70 S6 kinase activity. The activitiesof each PK are given relative to their activity towards Crosstide (100).The results are shown ±SEM for 2 experiments each carried out intriplicate.

Discussion

We have established that the minimum consensus sequence for efficientphosphorylation by RAC-PKα is Arg-Xaa-Arg-Yaa-Zaa-Ser-Hy, where Xaa isany amino acid, Yaa and Zaa are small amino acid other than Gly (Ser,Thr and Ala) and Hyd is a bulky hydrophobic residue (Phe and Leu). SeeTable 7.1. The heptapeptide with the lowest Km value was RPRTSSF (SEQ IDNO: 9), its Km of 5 μM being comparable to many of the best peptidesubstrates identified for other PKs. The Vmax for this peptide (250nmoles min-1 mg-1) may be an underestimate because the RAC-PKα wasobtained by immunoprecipitation from extracts of IGF-1 stimulated 293cells over-expressing this PK, and a significant proportion of theRAC-PKα may not have been activated by IGF-1 treatment.

The requirement for Arg residues at positions n-3 and n-5 (where n isthe site of phosphorylation) seems important, because substitutingeither residue with Lys decreases phosphorylation drastically. Ser andThr residues were preferred at positions n-1 and n-2, although the Kmvalue was only increased about 5-fold if both of these residues werechanged to Ala. Ser was preferred at position n, since changing it toThr caused a 6-fold increase in the Km. The peptide RPRAATF (SEQ ID NO:16), which was phosphorylated with a Km of 25 μM and similar Vmax toRPRTSSF (SEQ ID NO: 9), may therefore be a better substrate for assayingRAC-PKα in partially-purified preparations, because unlike Crosstide, itcontains only one phosphorylatable residue and is not phosphorylatedsignificantly by MAPKAP-K1 or p70 S6 kinase. See Table 7.2; FIG. 18; andsee below.

The Pro at position n-4 was not altered in this study because it wasalready clear that this residue was not critical for the specificity ofRAC-PKα. Residue n-4 is Pro in GSK3β but Ala in GSK3α. Both GSK3isoforms are equally good substrates for RAC-PKα in vitro [see Cross etal. (1995), supra], and the peptide GRARTSSFA (SEQ ID NO: 17),corresponding to the sequence in GSK3a, is phosphorylated by RAC-PKαwith a Km of 10 μM and Vmax of 230 U/mg. See Table 7.1A, peptide 2.Moreover, in histone H2B, the residue located 4 amino acids N-terminalto the RAC-PKα phosphorylation site is Serine. See FIG. 17. The presenceof Glu and Lys at positions n-1 and n-2 may explain why histone H2B isphosphorylated by RAC-PKα with a 4-fold lower Vmax than the peptideRPRTSSF (SEQ ID NO: 9).

Two other PKs which are activated by insulin and other growth factors,p70 S6 kinase and MAPKAP-K1, require basic residues at positions n-3 andn-5 [see Leighton et al. (1995), supra], explaining why they alsophosphorylate and inactivate GSK3 in vitro. See Sutherland, Leighton andCohen, Biochem J (1993), supra. Indeed, there is evidence that MAPKAP-K1plays a role in the inhibition of GSK3 by EGF because, unlike inhibitionby insulin which is prevented by inhibitors of PI 3-kinase, theinhibition of GSK3 by EGF is only suppressed partially by inhibitors ofPI 3-kinase. Moreover, in NIH 3T3 cells, the inhibition of GSK3α andGSK3β by EGF is largely prevented by expression of a dominant negativemutant of MAPKAP kinase-1. See Eldar-Finkelman, Seger, Vandenheede andKrebs (1995), supra. In contrast, p70S6 kinase is not rate limiting forthe inhibition of GSK3 in the cells that have been examined so farbecause rapamycin, which prevents the activation of p70 S6 kinase by EGFor insulin, has no effect on the inhibition of GSK3 by these agonists.See Cross et al. (1995), supra; and Saito, Vandenheede and Cohen,Biochem J (1994), supra.

Additional similarities between p70 S6 kinase, MAPKAP-K1 and RAC-PKαinclude the failure to phosphorylate peptides containing Pro at positionn+1 and dislike of a Lys at the same position. This suggests that, invivo, these kinases are unlikely to phosphorylate the same residues asMAP kinases (which phosphorylates Ser/Thr-Pro motifs) or PK C, whichprefers basic residues C-terminal to the site of phosphorylation.However, the present work has also revealed significant differences inthe specificities of these enzymes. In particular, MAPKAP-K1 and (to alesser extent) p70 S6 kinase can tolerate substitution of the Arg atposition n-5 by Lys, whereas RAC-PKα cannot. See Tables 7.1A and 7.2A;and Leighton et al. (1995), supra. MAPKAP-K1 and p70 S6 kinase can alsotolerate, to some extent, substitution of Arg at position n-3 by Lys.For example, the peptide KKRNKTLSVA is phosphorylated by MAPKAP-K1 andp70 S6 kinase with Km values of 17 μM and 34 μM, respectively, ascompared to Km values of 0.7 μM and 1.5 μM for the peptide KKRNRTLSVA(SEQ ID NO: 14). See Table 7.2A. In contrast, RAC-PKα does notphosphorylate the peptide KKRNKTLSVA (see Table 7.2A) or any otherpeptide that lacks Arg at position n-3. RAC-PKα and p70 S6 kinase, butnot MAPKAP-K1, phosphorylate Thr, as well as Ser (see Table 7.1 A) andcan phosphorylate peptides lacking any residue at position n+2 [seeLeighton et al. (1995), supra; and Table 7.2A], while RAC-PKα andNAPKAP-K1, but not p70 S6 kinase, can tolerate substitution of both then-1 and n-2 positions of the peptide RPRTSSF (SEQ ID NO: 9) with Ala.See Table 7.2B. These differences explain why the peptide RPRAATF (SEQID NO: 16) is a relatively specific substrate for RAC-PKα.

One of the best peptide substrates for MAPKAP-K1 and p70 S6 kinaseKKRNRTLSVA (SEQ ID NO: 14) was a poor substrate for RAC-PKα (see Table7.2, peptide 2), despite the presence of Arg at positions n-3 and n-5.The presence of Leu at position n-1 and Val at position n+1 are likelyto explain the high Km for phosphorylation, because RAC-PKα prefers asmall hydrophilic residue at the former position and a largerhydrophobic residue at the latter position. See Tables 7.1 and 7.2.

EXAMPLE 12

This example demonstrates that coexpression of GSK3 in 293 cells witheither the WT or a constitutively-activated RAC-PK results in GSK3becoming phosphorylated and inactivated. However coexpression of amutant of GSK3 in which Ser9 is mutated to an Ala residue is notinactivated under these conditions. These experiments provide furtherevidence that RAC-PKα activation can mediate the phosphorylation andinactivation of GSK3 in a cellular environment, and could be used as anassay system to search for specific RAC-PK inhibitors.

Monoclonal antibodies recognising the sequence EFMPME (EE) (SEQ ID NO:18) antibodies and the EQKLISEEDL (SEQ ID NO: 19) c-Myc purchased fromBoehringer, Lewis, UK.

Construction of expression vectors and transfections into 293 cells.HA-RAC-PKα, HA-KD-RAC-PK and 308D/473D HA-RAC-PKα as was describedpreviously. See Alessi et al. (1996), supra.

A DNA construct expressing human GSK3B with the EFMPME (EE) (SEQ ID NO:18) epitope tag at the N-terminus was prepared as follows. A standardPCR reaction was carried out using as a template the human GSK3β cDNAclone in the pBluescript SK+vector and the oligonucleotidesGCGGAGATCTGCCACCATGGAGTTCATGCCCATGGAGTCAGG GCGGCCCAGAACC (SEQ ID NO: 20)and GCGGTCCGGMCATAGTCCAGCACCAG (SEQ ID NO: 21) that incorporate a bgl IIsite (underlined) and a Bspe I site (double underlined). A 3-wayligation was then set up in which the resulting PCR product wassubcloned as a Bgl II-Bspe I fragment together with the C-terminal BspeI-Cla I fragment of GSK3β into the Bgl II-Cla I sites of the pCMV5vector. See Andersson et al., J Biol Chem, Vol. 264, No. 14, pp.8222-8229 (1989). The construct was verified by DNA sequencing and purified using the Quiagen plasmid Mega kit according to the manufacturersprotocol. The c-Myc GSK3, BA9 construct encodes GSK3β in which Ser9 ismutated to Ala and possesses a c-myc epitope tag at the C-terminus andwas prepared as described in Sperber, Leight, Goedert and Lee, NeurosciLett, Vol. 197, No. 2, pp. 149-153 (1995). The c-Myc GSK3β A9 gene wasthen subcloned into xba I/ECOR I sites of the pCMV5 eukaryoticexpression vector.

Cotransfection of GSK3β with RAC-PKα and its assay. 293 cells growing on10 cm diameter dishes were transfected with 10 μg of DNA constructsexpressing EE-GSK3, Myc-GSK3A9 in the presence or absence of HA-RAC-PK,HA-KD-RAC-PK or HA-308D/473D-RAC-PK exactly as described in Alessi etal. (1996), supra. The cells were grown in the absence of serum for 16hours prior to lysis, and then lysed in 1.0 mL of ice-cold buffer A (50mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (by vol) Triton X100, 1 mMsodium orthopervanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mMsodium pyrophosphate, 1 uM Microcystin-LR, 0.27 M sucrose, 1 mMbenzamidine, 0.2 mM phenylmethylsulphonyl fluoride, 10 μg/mL leupeptinand 0.1% (by vol) 2-mercaptoethanol). The lysate was centrifuged at 4°C. for 10 minutes at 13,000μg and an aliquot of the supernatant (100 μgprotein) was incubated for 30 minutes on a shaking platform with 5 μL ofProtein G-Sepharose coupled to lug of EE monoclonal antibody. Thesuspension was centrifuged for 1 minute at 13,000×g, the ProteinG-Sepharose-antibody-EE-GSK3β complex washed twice with 1.0 mL of bufferA containing 0.5 M NaCl, and three times with Buffer B (50 mM Tris, pH7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1% (by vol)2-mercaptoethanol), and the immunoprecipitate assayed for GSK3 activityafter incubation with either PP2A or microcystin inactivated PP2A asdescribed previously. See Cross et al. (1994), supra.

Results

Cotransfection of GSK3β with RAC-PKα in 293 cells results in GSK3phosphorylation and inactivation human embryonic kidney 293 cells weretransfected with a DNA construct expressing EE-epitope tagged GSK3βeither in the presence or absence of DNA constructs expressingWT-RAC-PKα, a catalytically inactive RAC-PKα or a constitutively activeHA-(308D/473D)-RAC-PKα. Cells were serum starved for 16 hours.Thirty-six (36) hours post-transfection, the cells were lysed, and theGSK3β immunoprecipitated from the lysates using monoclonal EE antibodiesand the GSK3β activity measured before and after treatment with PP2A.When EE-GSK3β was expressed alone or in the presence of a catalyticallyinactive RAC-PKα, treatment of the EE-GSK3β with PP2A only resulted inabout a 12% increase in activity. See FIG. 19 (a). However when EE-GSK3βwas coexpressed with either the WT RAC-PKα or the constitutivelyactivated 308D/473D-HA-RAC-PKα, treatment of the EE-GSK3 from these celllysates with PP2A resulted in a 68% and 85% increase in the GSK3activity, respectively. Coexpression of Myc-GSK3β A9 with HA-RAC-PK orthe constitutively active 308D/473D-HA-RAC-PKα did not result in anysignificant inactivation of this mutant of GSK3 as judged by its abilityto be reactivated by PP2A. See FIG. 19 (b). These data demonstrate thateven in a cellular environment, RAC-PKα is capable of phosphorylatingGSK3β at Ser9 and inactivation of the enzyme. To estimate the relativelevels of EE-GSK3β and RAC-PKα, EE-GSK3 and HA-RAC-PKα wereimmunoprecipitated from equal volumes of cell lysate, and theimmunoprecipitates run on an SDS-polyacrylamide gel, and the gel stainedwith Coomassie Blue. These experiments revealed that both the WTHA-RAC-PKα and the 308D/473D-RAC-PKα were expressed at a 20- to 30-foldhigher level than GSK3a, whereas KD-RAC-PKα is expressed at a level thatis about 5-fold lower than that of the WT RAC-PKα. Under the conditionsused for the immunoprecipitations, no RAC-PKα was co-immnuoprecipitatedwith GSK3β, or no GSK3β was co-immunoprecipitated with the RAC-PKα (datanot shown). Coexpression of EE-GSK3β with all forms of RAC-PKα resultedin about a 2- to 3-fold decrease in the level of expression on EE-GSK3βcompared to when it is expressed alone in cells.

EXAMPLE 13

Basic Assay for Identifying Agents which Affect the Activity of RAC-PK

A 40 μL assay mix was prepared containing PK (0.2 U/mL) in 50 mMTris/HCl, pH 7.5, 0.1 mM EGTA, 0.1% (by vol) 2-mercaptoethanol, 2.5 μMPKI, PK substrate (30 μM), and the indicated concentration of Ro-318220or GC 109203X (test inhibitors). After incubation on ice for 10 minutes,the reaction was started by the addition of 10 μL of 50 mM magnesiumacetate and 0.5 mM [γ³²P]ATP (100-200 cpm/pmol). For the assay of mixedisoforms of PKC 20 μM diacylglycerol, 0.5 mM CaCl₂ and 100 μMphosphatidylsene were also present in the incubations. The assays werecarried out for 15 minutes at 30° C., then terminated and analyzed asdescribed. See Alessi et al., Methods Enzymol, Vol. 255, pp. 279-290(1995). One unit of activity was that amount of enzyme that catalyzedthe phosphorylation of 1 nmol os substrate in 1 minute. The finalconcentration of DMSO in each assay was 1% (by vol). This concentrationof DMSO does not inhibit any of these enzymes. Mixed isoforms of PKCwere assayed using histone H1 as substrate, while MAPKAP-K1β and p70 S6kinase were assayed using the peptide KKRNRTLSVA (SEQ ID NO: 14). SeeLeighton et al. (1995), supra. PK B was assayed with the peptideGRPRTSSFAEG [9] (SEQ ID NO: 5) and MAPKAP-K2 was assayed with thepeptide KKLNRTLSVA (SEQ ID NO: 27). See Stokoe, Caudwell, Cohen andCohen, Biochem J, Vol. 296, Pt. 3, pp. 843-849 (1993). p42 MAP kinasewas assayed using MBP, and MAPKK-1 and c-Rafl were assayed as describedin Alessi et al., Methods Enzymol (1995), supra.

Results

Effect of Ro 318220 and GF 109203X on PKs activated by growth factors,cytokines and cellular stresses. The mixed isoforms of PKC were potentlyinhibited by Ro 318220, with an IC₅₀ of 5 nM in our assay. See FIG. 20(a). In contrast, a number of PKs activated by growth factors (c-Rafl,MAPKK-1 and p42 MAP kinase) and 1 PK that is activated by cellularstresses and proinflammatory cytokines (MAPKAP-K2) were not inhibitedsignificantly by Ro 318022 in vitro. See FIG. 20 (a). PK B, an enzymethat is activated in response to insulin and growth factors wasinhibited by Ro 318220 (IC₅₀ of 1 μM) (see FIG. 20 (b) similar to theIC₅₀ for PKα. However, to our surprise, MAPKAP-K1β an enzyme which liesimmediately downstream of p42 and p44 MAP kinases and which is activatedin response to every agonist that stimulates this pathway, was inhibitedby Ro 318220 even more potently than the mixed PKC isoforms (IC₅₀=3 nm).See FIG. 20 (b). The p70 S6 kinase, which lies on a distinct growthfactor-stimulated signalling pathway from MAPKAP-K1, was also potentlyinhibited by Ro 318220 (IC₅₀=15 nM). See FIG. 20 (b).

Similar results were obtained using GF 109203X instead of Ro 3318220. Asreported previously [see Toullec et al., J Biol Chem, Vol. 266, No. 24,pp.15771-15781 (1991)], GC 109203X inhibited the mixed isoforms of PKC(IC₅₀=30 nM) without inhibiting PK B (see FIG. 21) or c-Raf, MAPKK-1 andp42 MAP kinase (data not shown). However MAPKAP-K1 B and p70 S6 kinasewere potently inhibited by this compound with IC₅₀ values of 50 nM and100 nM, respectively. See FIG. 21.

General Materials and Methods

Tissue culture reagents, MBP, microcystin-LR, and IGF-1 were obtainedfrom Life Technologies Inc. (Paisley, UK), insulin from Novo-Nordisk(Bagsvaerd, Denmark), phosphate free Dulbecco's minimal essential medium(DMEM) from (ICN, Oxon, UK), Protein G-Sepharose and CH-Sepharose fromPharmacia (Milton Keynes, UK), alkylated trypsin from Promega(Southampton, UK), 4-vinylpyridine, wortmannin andfluroisothiocyanante-labelled antimouse IgG from goat from Sigma-Aldrich(Poole, Dorset, UK). Polyclonal antibodies were raised in sheep againstthe peptides RPHFPQFSYSASGTA (SEQ ID NO: 22), corresponding to the last15 residues of rodent RAC-PKα, and MTSALATMRVDYEQIK (SEQ ID NO: 23),corresponding to residues 352-367 of human MAPKAPkinase-2, and affinitypurified on peptide-CH-Sepharose. Monoclonal HA antibodies were purifiedfrom the tissue culture medium of 12CA5 hybridoma and purified bychromatography on Protein G-Sepharose. The peptide RPRHFPQFSYSAS (SEQ IDNO: 24), corresponding to residues 465478 of RAC-PKα, was synthesized onan Applied Biosystems 430A peptide synthesizer. cDNA encoding residues46-400 of human MAPKAP kinase-2 was expressed in E. coli as a GST fusionprotein and activated with p38/RK MAP kinase by Mr A. Clifton(University of Dundee) as described previously. See Ben-Levy et al.,EMBO J, Vol. 14, No. 23, pp. 5920-5930 (1995).

Monoclonal antibodies recognizing the HA epitope sequence YPYDVPDYA (SEQID NO: 25), Protein G-Sepharose and histone H2B were obtained fromBoehringer (Lewes, UK). MAPKAP kinase-1 [see Sutherland, Leighton andCohen, Biochem J (1993), supra] and p70 S6 kinases [see Leighton et al.(1995), supra] were purified from rabbit skeletal muscle and rat liver,respectively.

Construction of Expression Vectors

The pECE constructs encoding the human HARAC-PKα and kinase-dead (K179A)HA-KD-RAC-PKα have already been described. See Andjelkovic et al.(1996), supra. The mutants at Ser473 (HA-473A RAC-PKα and HA-473DRAC-PKα were created by PCR using a 5′ oligonucleotide encoding aminoacids 406-414 and mutating 3′ oligonucleotide encoding amino acids468-480, and the resulting PCR products subcloned as Celil-EcoRIfragment into pECE.HA-RAC-PKα. The Thr308 mutants (HA-308A RAC-PKα andHA308D RAC-PKα) were created by the 2-stage PCR technique [see No etal., Gene, Vol. 77, pp. 51 -59 (1989)] and subcloned as Notl-EcoRIfragments into pECE HA-RAC-PK. The double-mutant HA-308D/473D RAC-PK wasmade by subcloning the CelII-EcoRI fragment encoding 473D into pECEHA-308D RAC-PKα. For construction of cytomegalovinus-driven expressionconstructs, BglII-XbaI fragments from the appropriate pECE constructswere subcloned into the same restriction sites of the pCMVS vector. SeeAndersson et al. (1989), supra.

All constructs were confirmed by restriction analysis and sequencing andpurified using Quiagen Plasmid Maxi Kit according to the manufacturer'sprotocol. All oligonucleotide sequences are available upon request.

³²P-labelling of L6 myotubes and immunoprecipitation of PKRα. L6 cellswere differentiated into myotubes on 10 cm diameter dishes. See Hundalet al., Endocrinology, Vol. 131, pp. 1165-1171 (1992). The myotubes weredeprived of serum overnight in DMEM, washed three times in phosphatefree DMEM and incubated for a further 1 hour with 5 mL of this medium.The myotubes were then washed twice with phosphate free DMEM andincubated for 4 hours with carrier-free [³²P]orthophosphate (1 mCi/mL).Following incubation in the presence or absence of 100 nM wortmannin for10 minutes, the myotubes were stimulated for 5 minutes at 37° C. in thepresence or absence of 100 nM insulin and placed on ice. The medium wasaspirated, the myotubes washed twice with ice-cold DMEM buffer and thenlysed with 1.0 mL of ice-cold buffer A (50 mM Tris/HCl, pH 7.5, 1 mMEDTA, 1 mM EGTA, 1% (by vol) Triton X100, 1 mM sodium orthopervanadate,10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1μM Microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mMphenylmethylsulphonyl fluoride, 10 pg/mL leupeptin, and 0.1% (by vol)2-mercaptoethanol). The lysates were centrifuged at 4° C. for 10 minutesat 13,000×g and the supernatants incubated for 30 minutes on a shakingplatform with 50 μL of Protein G-Sepharose coupled to 50 μg of preimmunesheep IgG. The suspensions were centrifuged for 2 minutes at 13,000×gand the supernatants incubated for 60 mintues with 30 μL of ProteinG-Sepharose covalently coupled to 60 μg of RAC-PKα antibody. See Harlowand Lane, Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988). The ProteinG-Sepharose-antibody-RAC-PKα complex was washed eight times with 1.0 mLof buffer A containing 0.5 M NaCl, and twice with 50 mM Tris/HCl, pH7.5, 0.1 mM EGTA and 0.1% (by vol) 2-mercaptoethanol (buffer B).

Assay of immunoprecipitated RAC-PKα and protein determinations. Threealiquots of each immunoprecipitate (each comprising only 5% of the totalimmunoprecipitated RAC-PKα) were assayed for RAC-PKα activity towardsthe peptide GRPRTSS FAEG (SEQ ID NO: 5) as described previously. SeeCross et al. (1995), supra. One unit of activity was that amount whichcatalyzed the phosphorylation of 1 nmol of substrate in 1 minute.Protein concentrations were determined by the method of Bradford, AnalBiochem, Vol. 72, pp. 248-254 (1976).

Tryptic digestion of in vivo phosphorylated RAC-PKα. Theimmunoprecipitated RAC-PKα was added to an equal volume of 2% (by mass)SDS and 2% (by vol) 2-mercaptoethanol, and incubated for 5 minutes at100° C. Aftercooling to room temperature, 4-vinylpyridine was added to afinal concentration of 2% (by vol) and the mixture was incubated for 1hour at 30° C. on a shaking platform, followed by electrophoresis on a10% polyacrylamide gel. After autoradiography, the 60 kDa bandcorresponding to rat RAC-PKα was excised and the gel piece homogenizedin 5 vols of 25 mM N-ethylmorpholine HCl, pH 7.7, containing 0.1% (bymass) SDS and 5% (by vol) 2-mercaptoethanol. The suspension wasincubated for 1 hour at 37° C. on a shaking platform, then centrifugedfor 1 minute at 13,000×g and the supernatant collected. The pellet wasincubated for a further 1 hour with 5 vols of the same buffer andcentrifuged for 1 minute at 13,000×g. The 2 supernatants, containing80-90% of the ³²P-radioactivity, were combined, 0.2 vols of 100% (bymass) trichloroacetic acid added, and the sample incubated for 1 hour onice. The suspension was centrifuged for 10 minutes at 13,000×g, thesupernatant discarded and the pellet washed 5 times with 0.2 mL ofwater. The pellet was then incubated at 30° C. with 0.3 mL of 50 mMTris/HCl, pH 8.0, 0.1% (by vol) Triton X100 containing 1 μg of alkylatedtrypsin. After 3 hours, another 1 μg of trypsin was added and thesuspension left for a further 12 hours. Guanidinium hydrochloride (8 M)was added to bring the final concentration to 1.0 M in order toprecipitate any residual SDS and, after standing on ice for 10 minutes,the suspension was centrifuged for 5 minutes at 13,000×g. Thesupernatant containing 90% of the ³²P-radioactivity was chromatographedon a Vydac C18-column as described in the legend to FIG. 2.

Transfection of 293 cells and immunoprecipitation of HA-tagged RAC-PKα.Human embryonic kidney 293 cells were cultured at 37° C. in anatmosphere of 5% CO₂, on 10 cm diameter dishes in DMEM containing 10%fetal calf serum (FCS). Cells were split to a density of 2×10⁶ per 10 cmdish, and after 24 hours at 37° C., the medium was aspirated and 10 mLof freshly-prepared DM EM containing 10% FCS added. Cells weretransfected by a modified calcium phosphate method [see Chen andOkayama, Biotechniques, Vol. 6, No. 7, pp. 632-638 (1988)] with 1 μg/mLDNA per plate. Ten (10) μg of plasmid DNA in 0.45 mL of sterile waterwas added to 50 μL of sterile 2.5 M CaCl₂, and then 0.5 mL of a sterilebuffer composed of 50 mM N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonicacid/HCl, pH 6.96, 0.28 M NaCl and 1.5 mM Na₂HPO₄ was added. Theresulting mixture was vortexed for 1 minute, allowed to stand at roomtemperature for 20 minutes, and then added dropwise to a 10 cm dish of293 cells. The cells were placed in an atmosphere of 3% CO₂, for 16hours at 37° C., then the medium was aspirated and replaced with freshDMEM containing 10% FCS. The cells were incubated for 12 hornus at 37°C. in an atmosphere of 5% CO₂, and then for 12 hours in DMEM in theabsence of serum. Cells were preincubated for 10 minutes in the presenceof 0.1% DMSO or 100 nM wortmannin in 0.1% DMSO and then stimulated for10 minutes with either 100 nM insulin or 50 ng/mL IGF-1 in the continuedpresence of wortmannin. After washing twice with ice-cold DMEM the cellswere lysed in 1.0 mL of ice-cold buffer A, the lysate was centrifuged at4° C. for 10 minutes at 13,000×g and an aliquot of the supernatant (10μg protein) was incubated for 60 minutes on a shaking platform with 5 μLof Protein G-Sepharose coupled to 2 μg of HA monoclonal antibody. Thesuspension was centrifuged for 1 minute at 13,000×g, the ProteinG-Sepharose-antibody-HA-RAC-PKα complex washed twice with 1.0 mL ofbuffer A containing 0.5 M NaCl, and twice with buffer B, and theimmunoprecipitate assayed for RAC-PKα activity as described above.

³²P-Labelling of 293 Cells Transfected with HA-RAC-PKα

Two hundred ninety-three (293) cells transfected with HA-RAC-PKα DNAconstructs. were washed with phosphate-free DMEM, incubated with [³²p]orthophosphate (1 mCi/mL) as described for L6 myotubes, then stimulatedwith insulin or IGF1 and lysed, and RAC-PKα immunoprecipitated asdescribed above. The NP-labelled HA-RAC-PKα immunoprecipitates werewashed, alkylated with 4-vinylpyridine, electrophoresed and digestedwith trypsin as described above for the endogenous RAC-PKα present inrat L6 myotubes.

Transfection of COS-1 Cells and Immunoprecipitation of HA-RAC-PKα

COS-1 cells were maintained in DMEM supplemented with 10% FCS at 37° C.in an atmosphere of 5% CO₂. Cells at 70-80% confluency were transfectedby a DEAE-dextran method [see Seed and Aruffo, Proc Natl Acad Sci USA,Vol. 84, pp. 3365-3369 (1987)], and 48 hours later serum-starved for 24hours. Cells were lysed in a buffer containing 50 mM Tris-HCl, pH7.5,120 mM NaCl, 1% Nonidet P-40, 25 mM NaF, 40 mM sodium-,β-glycerophosphate, 0.1 mM sodium orthopervanadate, 1 mM EDTA, 1 mMbenzamidine, 1 mM phenylmethylsulphonyl fluoride and lysates centrifugedfor 15 minutes at 13,000×g at 4° C. Supernatants were pre-cleared oncefor 30 minutes at 4° C. with 0.1 vols of 50% Sepharose 4B/25% Pansorbin(Pharmacia and Calbiochem, respectively) and HA-RAC-PKαimmunoprecipitated from 1 mg of extract using the 12CA5 antibody coupledto Protein A Sepharose beads. Immunoprecipitates were washed twice withlysis buffer containing 0.5 M NaCl and once with lysis buffer.

Immunoblotting and Quantification of Levels of PKα Expression.

Cell extracts were resolved by 7.5% SDS-PAGE and transferred toImmobilon membranes (Millipore). Filters were blocked for 30 minutes ina blocking buffer containing 5% skimmed milk in 1×TBS, 1% Triton X-100and 0.5% Tween 20, followed by a 2 hours incubation with the 12CA5supernatant 1000-fold diluted in the same buffer. The secondary antibodywas alkaline (Alk) conjugated anti-mouse Ig from goat (SouthernBiotechnology Associates, Inc), 1000-fold diluted in the blockingbuffer. Detection was performed using AP color development reagents fromBio-Rad according to the manufacturer's instructions. Quantification oflevels of RAC-PKα expression was achieved by chemiluminescence, usingfluroisothiocyanante-labelled antimouse IgG from goat as the secondaryantibody and the Storm 840/860 and ImageQuant software from MolecularDynamics.

All peptides used to assay RAC-PKα, and TTYADFIASGRTGRRNAIHD (SEQ ID NO:26), the specific peptide inhibitor of cyclic AMP dependent PK—PKI, weresynthesized on an Applied Biosystems 431A peptide synthesizer. Theirpurity (>95%) was established by HPLC and electrospray massspectrometry, and their concentrations were determined by guantitativeamino acid analysis.

Preparation and Assay of RAC-PKα

The construction of cytomegalovirus vectors (pCMV5) of the human HAepitope-tagged WT (HA-RAC-PKα) was described previously. See Alessi etal. (1996), supra. Two hundred ninety-three (293) cells grown on 10 cmdishes were transfected with a DNA construct expressing HA-RAC-PKα usinga modified calcium phosphate procedure. See Alessi et al. (1996), supra.The cells were deprived of serum for 16 hours prior to lysis and, whereindicated, were stimulated for 10 minutes in the presence of 50 ng/mLIGF-1 to activate RAC-PKα. The cells were lysed in 1.0 mL ice-coldbuffer A (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (by vol)Triton X-100, 1 mM sodium orthovanadate, 10 mM sodiumβ-glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 μMMicrocystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mMphenylmethylsulphonyl fluoride, 10 μg/mL leupeptin and 0.1% (by vol)2-mercaptoethanol) the lysate centrifuged at 4° C. for 10 minutes at13,000×g and the supernatant obtained from one 10 cm dish of cells (2-3mg protein) was incubated for 60 minutes on a shaking platform with 20p1 of Protein G-Sepharose coupled to 10 μg of HA monoclonal antibody.The suspension was centrifuged for 1 minute at 13,000×g, the ProteinG-Sepharose-antibody-HA-RAC-PKα complex washed twice with 1.0 mL ofbuffer A containing 0.5 M NaCl, and twice with buffer B (50 mM Tris/HCl,pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1% (by vol)2-mercaptoethanol). The RAC-PKα immunoprecipitates were diluted inbuffer B to an activity of 2.0 U/mL towards the Crosstide peptideGRPRTSSFAEG (SEQ ID NO: 5) and 0.1 mL aliquots snap frozen in liquidnitrogen and stored at −80° C. No significant loss of RAC-PKα activityoccurred upon thawing the RAC-PKα immunoprecipitates or during storageat −80° C. for up to 3 months. The standard RAC-PKα_assay (50 μL)contained: 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.1% (by vol)2-mercaptoethanol, 2.5 μM PKI, 0.2 U/ml RAC-PKα, Crosstide (30 μM), 10mM magnesium acetate and 0.1 mM [Y³²P]ATP (100-200 cpm/pmol). The assayswere carried out for 15 minutes at 30° C., the assay tubes beingagitated continuously to keep the immunoprecipitate in suspension, thenterminated and analyzed as described. See Alessi et al. (1995), supra.One unit of activity was that amount of enzyme which catalyzed thephosphorylation of 1 nmol of Crosstide in 1 minute. The phosphorylationof other peptides, histone H2B and MBP were carried out in an identicalmanner. All the Crosstide activity in HA-RAC-PKα immunoprecipitates iscatalysed by RAC-PKα (see Results) and the RAC-PKα concentration in theimmunoprecipitates was estimated by densitometric scanning of Coomassieblue-stained polyacrylamide gels, using bovine serum albumin as astandard. Protein concentrations were determined by the method ofBradford using bovine serum albumin as standard. See Bradford (1976),supra. Michaelis constants (Km) and Vmax values were determined fromdouble reciprocal plots of 1/V against 1/S, where V is the initial rateof phosphorylation, and S is the substrate concentration. The standarderrors for all reported kinetic constants were within <±20%, and thedata is reported as mean values for 3 independent determinations. FIG.16 shows the results relative to those obtained for unstimulatedRAC-PKα.

Tryptic Digestion of Histone 2B Phosphorylated by RAC-PKα

Histone H2B (30 μM) was phosphorylated with 0.2 U/mL HA-RAC-PKα. After60 minutes, 0.2 vol of 100% (by mass) trichloroacetic acid was added,and the sample incubated for 1 hour on ice. The suspension wascentrifuged for 10 minutes at 13,000×g, the supernatant discarded andthe pellet washed 5 times with 0.2 mL of ice-cold acetone. The pelletwas re-suspended in 0.3 mL of 50 mM Tris/HCl, pH 8.0, 0.1% (by vol)reduced Triton-X100 containing 2 pg of alkylated trypsin and, afterincubation for 16 hours at 30° C., the digest was centrifuged for 5minutes at 13,000×g. The supernatant, containing 95% of the³²P-radioactivity, was chromatographed on a Vydac C18-columnequilibrated with 0.1% (by vol) TFA in water. With reference to theresults shown in FIG. 17, the columns were developed with a linearacetonitrile gradient (diagonal line) at a flow rate of 0.8 mL/min. andfractions of 0.4 mL were collected.

(a) Tryptic peptide map of ³²P-labelled histone H2B, 70% of theradioactivity applied to the column was recovered from the major³²P-peptide eluting at 19.5% acetonitrile.

(b) A portion of the major ³²P-peptide (50 pmol) was analyzed on anApplied Biosystems 476A sequencer, and the Pth amino acids identifiedafter each cycle of Edman degradation are shown using the single-lettercode for amino acids. A portion of the major ³²P-peptide (1000 cpm) wasthen coupled covalently to a Sequelon arylamine membrane and analyzed onan Applied Biosystems 470A sequencer using the modified program. SeeStokoe et al. (1992), supra. ³²P-radioactivity was measured after eachcycle of Edman degradation. TABLE 7.1 Molecular basis for the substratespecificity of RAC-PKα Peptides Km (μM) Vmax (U/mg) V (0.1 mM) A  1.GRPRTSSFAEG 4 250 100  2. RPRTSSFA 8 305 109  3. GRPRTSSF 8 385 129  4.RPRTSSF 5 260 105  5. RPRTS T F 30 243 78  6. RPRTS A F — 0  7. PRTSSF —0  8. RPRTSS >500 ND 2  9. KPRTSSF >500 ND 4 10. RPKTSSF >500 ND 2 B  1.RPRTSSF 5 260 105  2. RPRTSS L 8 278 104  3. RPRTSS V 21 300 102  4.RPRTSS A 250 265 30  5. RPRTSS K 80 308 67  6. RPRTSS E >500 ND 9  7.RPRTSS PA* — 0 C  1. RPRTSSF 5 260 105  2. RPRASSF 12 230 89  3. RPRVSSF25 273 77  4. RPRGSSF 60 163 37  5. RPRNSSF >500 ND 21  6. RPRTA SF 20213 83  7. RPRTG SF 25 233 77  8. RPRTV SF 30 365 89  9. RPRTN SF 30 30081 10. RPRAA SF 25 215 77 11. RFRGG SF 105 345 55 12. RPRGA SF 105 16037 13. RPRAG SF 49 114 70The phosphorylated residue is shown in boldface typeThe altered residue is underlined.V(100 μM) is the relative rate of phosphorylation at 0.1 mM peptiderelative to peptide 1.ND = not determined.*An alanine residue was added to the C-terminal of the peptide RPRTSSP,since we have experienced difficulty in synthesizing peptidesterminating in Pro.

TABLE 7.2. Comparison of the Substrate Specificities of RAC-PKα, MAPKAPKinase-1 and p70S6 Kinase Protein MAPKAP kinase Bα kinase-1 p70 S6kinase K_(m) V_(max) K_(m) V_(max) Km V_(max) Peptide (mM) (U/mg) (mM)(U/mg) (mM) (U/mg) A 1. KKKNRTLSVA 185 270 0.2* 1550*  33*  890* 2.KKRNRTLSVA 80 300 0.7* 1800* 1.5* 1520* 3. KKRNKTLSVA >500 ND  17*  840* 34*  760* 4. KKRNRTLTV 388 330  40*  270* 4.8* 1470* B 1. GRPRTSSFAEG 4250 2  790  3 1270  2. RPRTSSF 5 260 12  840  125  705  3. RPRTSTF 30240 >500 ND 211  590  4. RPRAASF 25 215 20 1020  >500 ND 5. RPRAATF 25230 >500 ND >500 NDPeptides 1 and 2 are very good substrates for MAPKAP kinase-1 and p70 S6kinase, and Peptide 3 is a relatively specific substrate for p70 S6kinase.*Data reported previously.ND = not determined.

EXAMPLE 14

Mitogenic Stimulation and Phosphorylation of RAC-PK

The Swiss 3T3 cell line [see {haeck over (S)}u{haeck over (s)}a andThomas, Proc Natl Acad Sci USA, Vol. 87, pp. 7040-7044 (1990)] isutilized to investigate the possible involvement of RAC-PK in growthfactor signalling. Quiescent Swiss 3T3 cells are serum-starved for 24hours, followed by stimulation with 10% FCS.

(a) Kinase activity is assessed by immunoprecipitating RAC-PK andassaying the kinase using MBP as a substrate. Briefly, cell freeextracts are prepared by scraping preconfluent cells into ice-cold TBS,lysing the cells in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mMEDTA, 1.0% Triton X-100, 2 mM EGTA, 1 mM PMSF, 20 μM leupeptin, 20 μMaprotenin and 10 μM molybdate. Lysates are centrifuged for 15 minutes at12,000×g at 4° C. RAC-PKα is immunoprecipitated from pasorbin-clearedextracts using a rabbit polyclonal antibody specific for the conservedC-terminus (anti-RAC⁴⁶⁹⁻⁴⁸⁰) [see Jones et al. (1991), supra] raised byinjecting rabbits subcutaneously with the peptide FPQFSYSASSTA (SEQ IDNO: 7) coupled to keyhole limpet haemocyanin and purified byprecipitation using 50% (NH₄)₂SO₄ followed by affinity chromatography onRAC-PK coupled Affigel® 10 column (Bio-Rad). These antisera alsorecognize the b/AKT2 isoform, because its C-terminus differs from thatof RAC-PKα in the last 3 amino acids. RAC-PK activity is assayed asdescribed previously using MBP as substrate. See Jones et al. (1991),supra. The extracts are incubated for 2 hours at 4° C. with theantiserum (2 μg/100 μL extract) the immunoprecipitates collected usingProtein A sepharose and washed with lysis buffer. The protein sepharosebeads are resuspended in 100 μL of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 10pm molybdate and 35 μL used for the kinase assay, as follows.

Reaction mixtures in a final volume of 50 μL contain 50 mM Tris-HCl, pH7.5, 10 mM MgCl₂, 1 mM DTT, 1 mM PK inhibitor, PKI peptide, 25 pg of MBP(Sigma), 50 μM (γ-32P) ATP (3500 cpm/pmol) and 35 μL ofimmunoprecipitate from cell free extracts or purified fractions ofRAC-PK. After incubation at 30° C. for 10, 30 or 60 minutes samples areanalyzed by 12% SDS/PAGE followed by autoradiography and quantified byscintillation counting of the phosphorylated MBP bands.

Immunoprecipitated RAC-PK activity is found to be 2- to 4-fold higher inserum-stimulated cells versus quiescent cells. Activation occurs within5 minutes and kinase activity remains elevated for at least 120 minutes.

(b) Activation coincides with decreased mobility of RAC-PK on SDS-PAGE.In order to determine which forms are present on SDS-PAGE gels,immunoblotting is performed using anti-RAC-PK antisera prepared asabove. Cell extracts and immunoprecipitates are resolved by 7.5%SDS-PAGE, transferred to Immobilon-P membranes (Millipore) and incubatedwith the anti-RAC⁴⁶⁹⁻⁴⁸⁰ antibody. Detection is performed using Alkphosphatase-conjugated anti-rabbit antibody.

At least 3 different forms can be detected by immunoblot analysis,termed a, b and c. The kinase from quiescent cells migrates as a doubletof the a and b forms and during stimulation a slower migrating form cappears, followed by disappearance of form a. These results suggest thatRAC-PK activity is modulated by reversible phosphorylation.

(c) To test this possibility the in vivo effects of phosphataseinhibitors okadaic acid and vanadate on RAC-PK from Swiss 3T3 cells areexamined. Cells are serum-starved for 24 hours, followed by stimulationwith 1 μM okadaic acid, or 0.1 mM vanadate prepared with 0.1 mM H₂O₂[see Posner et al., J Biol Chem, Vol. 269, pp. 4596-4604 (1994)],optionally in conjunction with 10% FCS. Treatment of cells with okadaicacid, a specific inhibitor of PP2A and PP1, induces a 3-fold increase inRAC-PK activity and decreases electrophoretic mobility. Simultaneoustreatment with 1 μM okadaic acid and 10% serum causes a 5-foldactivation and a larger alteration of the electrophoretic mobility. An11-fold activation is observed following treatment with 0.1 mM vanadate,which converts the major part of the protein into the slowest-migratingform c.

In order to confirm that multiple electrophoretic mobility forms reflectdifferent phosphorylation states of the kinase, RAC-PK isimmunoprecipitated from ³²P-labelled quiescent and vanadate-stimulatedSwiss 3T3 cells. Swiss 3T3 cells are arrested in phosphate-free DMEM/FCSas described [see {haeck over (S)}u{haeck over (s)}a and Thomas (1990),supra] and serum-starved for 16 hours prior to labelling with[³²P]orthophosphate for 6-10 hours (2 mCi per 15 cm dish). Stimulationis performed 0.1 mM vanadate. Quantification of phosphorylation isperformed using the ImageQuant software. Vanadate treatment leads to a3- to 4-fold increase in phosphorylation, demonstrating that themobility forms b and c represent phosphorylated RAC-PK.

(d) In order to determine which residues are phosphorylated in activatedRAC-PK, phosphoamino acid analysis is carried out on cells labelled asabove according to Boyle et al., Methods Enzymol, Vol. 201, pp. 110-149(1991). The kinase from arrested cells appears phosphorylated mainly onSer residues, and at low levels on Thr, with a ratio of 12:1. Vanadatestimulation leads to an increase in phosphoserine and, in particular, inphosphothreonine content, reducing the ratio to 4:1. Phosphotyrosine isnot detected after vanadate stimulation, either by phosphoamino acidanalysis, or by immunoblot analysis using an anti-phosphotyrosineantibody. These results show that RAC-PK is activated by aphosphorylation mechanism. Furthermore, we conclude that RAC-PKactivation mediated by vanadate is probably indirect, since vanadate isknown to be an inhibitor of tyrosine phosphatases.

EXAMPLE 15

Inactivation of RAC-PK by Protein Phosphatase 2A In Vitro

To confirm that RAC-PK is regulated by phosphorylation the effects ofPP2A treatment on the kinase immunoprecipitated from quiescent andvanadate-stimulated Swiss 3T3 cells are investigated. As treatment ofcells with 1 mM okadaic acid for 2 hours preferentially inactivates PP2Arather than PP1, RAC-PK is incubated either with the purified PP2Acatalytic subunit (PP2Ac), or PP2A dimer consisting of the catalytic andregulatory PR65 subunit (PP2A₂).

Immunoprecipitated RAC-PK is incubated with 0.3 U/mL of porcine musclePP2Ac or 1.7 U/mL of rabbit muscle PP2A₂ in 30 mL buffer containing 50mM Tris-HCl, pH 7.5, 1% b-mercaptoethanol, 1 mM MnCl₂, 1 mM benzamidineand 0.5 mM phenylmethylsulfonyl fluoride at 30° C. for 60 minutes (1 Uis defined as 1 nmol of Pi released from phosphorylase a per min.). Thereactions are stopped by addition of 50 nM calyculin A. The immunecomplexes formed are washed with 50 mM Tris-HCl, pH 7.5, 1 mMbenzamid,ine, 0.5 mM phenylmethylsulfonyl fluoride and 50 nM calyculin Aand RAC-PK is assayed as described above.

Dephosphorylation of the activated RAC-PK in vitro by PP2Ac results inan 84% reduction of kinase activity and concomitant change inelectrophoretic mobility, converting it from form c to b. PP2A₂treatment leads to a 92% reduction of activity and restores the proteinmobility on SDS-PAGE to the a/b doublet. These results confirm that theactivity changes observed are achieved by a reversible phosphorylationmechanism. Moreover, PP2A is indicated as a potential regulator ofRAC-PK activity in vivo.

EXAMPLE 16

RAC-PKα Stimulates p70^(s6k) Activity

In Swiss 3T3 cells RAC-PK is activated by insulin (4.5-fold), comparableto levels detected for p70^(s6k). In contrast, insulin has little or noeffect on p42^(mapk) and p44^(mapk) in these cells, suggesting thatRAC-PK and p70^(s6k) may reside on the same signalling pathway, which isa different pathway to the MAPK pathway.

In order to investigate this possibility the effects of wortmannin andrapamycin on serum induced activation of the two kinases is examined.Wortmannin, an inhibitor of PI 3-kinase, and immunosuppressant rapamycinblock the activation of p70^(s6k) by affecting the same set ofphosphorylation sites.

Stimulation of quiescent Swiss 3T3 fibroblasts leads to a ˜4-foldinduction of RAC-PK activity, whereas wortmannin treatment precedingserum stimulation almost completely blocks the activation. On the otherhand, rapamycin pretreatment does not exert any significant effect onRAC-PK activation. Wortmannin also blocks the appearance of slowestRAC-PK mobility form that is observed following serum treatment, whilerapamycin does not affect RAC-PK mobility. In the same experimentwortmannin and rapamycin pretreatment abolish p70s6k activation.

These results suggest that RAC-PK may lie upstream of p70^(s6k) on thep70^(s6k) signalling pathway, which is inhibited upstream of RAC-PK bywortmannin and downstream thereof by rapamycin. To examine thispossibility, the regulation of p70^(s6k) is investigated in a transientcotransfection assay using human 293 cells. RAC-PKα constructs areprepared by ligating the RAC-PKα cDNA [see Jones et al. (1991), supra]in-frame to the initiator methionine, in the mammalian expression vectorpECE. The construct is also subcloned into a CMV promoter-drivenexpression vector. The construct is confirmed by restriction analysisand sequencing. Constructs expressing Myc-tagged p70^(s)6k are obtainedfrom Dr. G. Thomas, Friederich Miescher Institut, Basel, Switzerland.Constructs are transfected into COS cells using standard procedures.Coexpression of RAC-PKα with p70^(s6k)-MyC results in a 3.5- and 3-foldincrease of basal and insulin-stimulated p70^(s6k)-Myc activity,respectively.

1. A method of treating a disease related to glycogen metabolism and/orprotein synthesis comprising administering a composition of RAC-PK, itsanalogues, isoforms, inhibitors, activators and/or the functionalequivalents thereof to a mammal in need thereof.
 2. The method of claim1 for the treatment of disease states where glycogen metabolism and/orprotein synthesis exhibits abnormality.
 3. The method of claim 1,wherein the disease related to glycogen metabolism is diabetes.
 4. Themethod of claim 1, wherein the disease related to protein synthesis iscancer.
 5. The method of claim 4, wherein the cancer is breast,pancreatic or ovarian cancer.
 6. The method of claim 1, wherein RAC-PKis RAC-PKα, β or γ, an analogue, isoform, inhibitor, activator or afunctional equivalent thereof.
 7. The method of claim 6, wherein RAC-PK,its analogue, isoform or functional equivalent is modified at one orboth of amino acids 308 and 473 by phosphorylation and/or mutation.
 8. Apharmaceutical composition comprising RAC-PK, its analogues, isoforms,inhibitors, activators and/or the functional equivalents thereof.
 9. Apeptide comprising an amino acid sequenceArg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyd, where Xaa is any amino acid, Yaa andZaa are any amino acid, and Hyd is a large hydrophobic residue, or afunctional equivalent of such a peptide.
 10. The peptide of claim 9,wherein Hyd is Phe or Leu, or a functional equivalent thereof.
 11. Apeptide as claimed in claim 9, wherein Yaa or Zaa or both are an aminoacid other than glycine.
 12. A peptide as claimed in claim 9, having theamino acid sequence as set forth in SEQ ID NO: 5, or a functionalequivalent thereof.
 13. A method of identifying agents able to influencethe activity of GSK3, said method comprising: (a) exposing a testsubstance to a substrate of GSK3; and (b) detecting whether saidsubstrate has been phosphorylated.
 14. A method of identifying agentswhich influence the activity of RAC-PK, comprising: (a) exposing a testsubstance to a sample containing RAC-PK, to form a mixture; and (b)exposing said mixture to the peptide of claim
 9. 15. The method of claim14, comprising the additional step of detecting whether said peptide hasbeen phosphorylated.
 16. The method of claim 15, wherein thephosphorylation state(s) of one or both of amino acids 308 and 473 onRAC-PK is determined.
 17. The method of claim 13, wherein the testsubstance is an analogue, isoform, inhibitor or activator of RAC-PK. 18.The method of claim 13, wherein steps (a) or (b), or both, are carriedout in the presence of divalent cations and ATP.
 19. An agent capable ofinfluencing the activity of RAC-PK, its isoforms, analogues and/orfunctional equivalents, by modifying amino acids 308 and/or 473 byphosphorylation or mutation.
 20. A method of determining the ability ofa substance to affect the activity or activation of RAC-PK, the methodcomprising exposing the substance to RAC-PK and phosphatidyl inositolpolyphosphate and determining the interaction between RAC-PK and thephosphatidyl inositol polyphosphate.
 21. A method of determining theability of a substance to combat diabetes, cancer or any disorder whichinvolves irregularity of protein synthesis or glycogen metabolism, themethod comprising exposing the substance to RAC-PK and phosphatidylinositol polyphosphate and determining the interaction between RAC-PKand the phosphatidyl inositol polyphosphate.
 22. The method of claim 20,wherein the interaction between RAC-PK and the phosphatidyl inositolpolyphosphate is measured by assessing the phosphorylation state ofRAC-PK.
 23. The method of claim 22, wherein the phosphorylation state ofRAC-PK at Thr308 and/or Ser473 is assessed.
 24. A method of identifyingactivators or inhibitors of GSK3 comprising exposing the substance to betested to GSK3 and determining the state of activation of GSK3.
 25. Amethod as claimed in claim 24, wherein the state of activation of GSK3is determined by assessing its phosphorylation.
 26. A method ofdetermining the suitability of a test substance for use in combattingdiabetes, cancer or any disorder which involves irregularity of proteinsynthesis or glycogen metabolism, the method comprising exposing thesubstance to be tested to GSK3 and determining the state of activationof GSK3.
 27. A method for screening for inhibitors or activators ofenzymes that catalyze the phosphorylation of RAC-PK, the methodcomprising exposing the substance to be tested to one or more enzymesupstream of RAC-PK and nucleoside triphosphate and determining whether(and optionally to what extent) the RAC-PK has been phosphorylated onThr308 and/or Ser473.
 28. A method for screening potential modulators ofinsulin-mediated intracellular signalling comprising the steps of: (a)incubating RAC-PK or a fragment thereof with the compound to bescreened; and (b) detecting interaction between the compound and RAC-PKor its fragment.
 29. The method according to claim 28, wherein RAC-PK isactivated.
 30. Method according to claim 28, wherein the RAC-PK fragmentis selected from the PH domain, the catalytic domain and the C-terminaldomain.
 31. A modulator of insulin-mediated intracellular signallingwhen identified by a method according to claim
 28. 32. A modulatoraccording to claim 31, which is selected from the group consisting ofIMPDH, GSK-3 and a polypeptide comprising SEQ ID NO:
 1. 33. A kitcomprising: (a) RAC, or a fragment thereof; (b) means for incubatingRAC-PK or its fragment with a compound to be screened; and (c) means fordetecting an interaction between RAC-PK or its fragment and thecompound.
 34. A RAC-PK polypeptide which is activated by effecting oneor both of the mutations Thr308→D and Ser473→D.
 35. A process forproducing an active kinase of a signalling pathway comprising treatmentthereof with a phosphatase inhibitor.
 36. A process according to claim35, which is carried out in vitro and comprises the steps of: (a)incubating together a kinase of a signalling pathway (b) an agentcapable of phosphorylating the kinase in order to activate it and aphosphatase inhibitor; and (c) purifying the kinase from the incubationmixture.
 37. A process according to claim 36, wherein thephosphorylating agent is a kinase of the signalling pathway which iscapable of phosphorylating the kinase of interest, thereby activatingit.
 38. A process according to claim 35, which is performed in cellswhich contain kinases of the signalling pathway.
 39. A process forscreening candidate modulators of a signalling pathway comprising: (a)incubating together a kinase of a signalling pathway and a phos phataseinhibitor; (b) adding the candidate signalling pathway modulator; and(c) determining the activity of the kinase.
 40. A process according toclaim 39, wherein steps (a) and (b) are performed contemporaneously. 41.A process according to claim 39, wherein the phosphatase inhibitor isokadaic acid.
 42. The process of claim 39, wherein the phosphataseinhibitor is vanadate.
 43. The process of claim 39, wherein the kinaseof the signalling pathway is RAC-PK.
 44. The process of claim 39,wherein the signalling pathway is an insulin-dependent signallingpathway.